Mass Spec 2 D Sample Handbook

Mass Spec Sample Handbook Featuring Thermo Scientific Pierce® Products to extract, separate, purify, detect, and quantify proteins

Table of Contents

Thermo Scientific Products for Mass Spectrometry Introduction

1

An Introduction to Mass Spectrometry

2

Cells and Tissue

3–5

SILAC Reagents Cell Surface Protein Isolation Kit

3–4 5

Cell Lysis and Protein Extraction

6–11

Cell Lysis Reagents Halt™ Protease Inhibitors Halt Phosphatase Inhibitor Protein Stabilizing Cocktail High Abundant Proteins SwellGel™ Blue Albumin Removal Kit Pierce IgG/Albumin Removal Kits

6–7 8–9 10 11 12–13 12 13

Pre-Fractionation and Enrichment

14–22

Phosphoprotein Enrichment Kit Glycoprotein Isolation Kits Ubiquitin Enrichment Kits Organelle Enrichment Kits: Lysosome, Perixisome, Nuclei Mitochondria Isolation Kits

14–15 16 17 18–19

Sample Clean-Up

23–28

Slide-A-Lyzer ® Dialysis Unit and Cassettes Zeba™ Desalt Spin Columns Pierce SDS-PAGE Sample Prep Kit

23–24 25 26

Gel Separation and Protein Detection

27–32

20–22

In-Gel Detection

34–35

In-Gel Tryptic Digestion Kit and Pierce C-18 Spin Columns

34–35

In-Solution Digestion

36–37

In-Solutions Tryptic Digestion and Guanidation Kit

36–37

Peptide Enrichment

38

Phosphopeptide Isolation Kit

38

Peptide Clean-Up

39

Pierce Strong Cation and Anion Ion Exchange Spin Columns

39

Mass Spectrometry

40

Ion Pairing Reagents Crosslinking Technical Handbook

40 40

Mass Spectrometry Applications

41–43

Crosslinking Reagents for the Analysis of Protein Interactions by Mass Spectrometry Analysis Gain new insights into protein interaction and folding

27 28–29 30 31 32

Protein Digestion

33

GelCode® Blue Stain Reagent

33

42

Protein Quantification by Mass Spectrometry

44–46

TMT Isobaric Mass Tagging Kits Custom HeavyPeptides™ BASIC and AQUA Kits Modified HeavyPeptides for Quantification of Phosphorylated Proteins

44 45

Instruments for Mass Spectrometry ™

SuperSignal® Chemiluminescent Substrates 2-D Sample Prep Kits 2-D Protein Markers Imperial™ Protein Stain Pierce Silver Stain for Mass Spectrometry

41

LTQ Orbitrap XL ETD MALDI LTQ XL TSQ Quantum Ultra

46 47-48 47 48 48

Introduction Introduction to Sample Preparation for 2-D and Mass Spectrometry Proper sample preparation means better results. Mass spectrometry (MS) is a powerful and versatile analytical tool that is playing an increasingly vital role in proteomics, metabolomics, small-molecule synthesis, and the drug discovery process. MS enables identification and quantification of known and unknown compounds by revealing their structural and chemical properties. Proper sample preparation for MS-based analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation for downstream analysis significantly impacts the separation and identification capabilities of MS instruments. With all of its forms of ionization and detection (e.g., ESI, MALDI, APcI, FT-MS, ion trap, TOF, Quad), MS allows for the analysis of samples ranging in mass from 50-300,000 daltons in low femtomole to high nanomole quantities. Because the proteome is so complex, there is no one standard method for preparing protein samples for MS analysis. Protocols differ depending on sample type, experiment and method of analysis. For example, preparing samples from a biological fluid involves a different set of procedures than those used for cell culture or tissue. Many factors are considered, including source, type, physical properties abundance, complexity, matrix effects and cellular location of the proteins.

Proteins of interest to biological researchers are usually part of a very complex mixture of other proteins. This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal amounts of constituents, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to “drown” or suppress signals from less abundant ones. The second problem is that the mass spectrum from a complex mixture is very difficult to interpret because of the overwhelming number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large number of peptide products. To contend with these problems, scientists for the Pierce Product Line, now part of the Thermo Scientific brand portfolio of products have developed a complete workflow of sample preparation solutions design for better MS analysis. Our researchers understand the need for integrated proteomics solutions and work to develop products and technologies that are compatible and supportive of MS analysis. The following workflow highlights options for preparing samples from a variety of starting materials for successful MS analysis.

Sample ionization Preparation of protein for MS analysis can be accomplished by many methods, however the initial principle of ion addition to the protein or peptide is essential for MS analysis. First, intact proteins are ionized by one of several techniques, and then introduced to a mass analyser. In the second, proteins are enzymatically digested into smaller peptides using a protease such as trypsin. Sample preparation is easier once whole proteins have been digested into smaller peptide fragments. Subsequently these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. This latter approach uses identification at the peptide level from known peptide sequences in databases to infer the existence of proteins.

Outside the United States, contact your local branch office or distributor.

1

Introduction

Thermo Scientific SILAC Cell Surface Biotinylation Cell Lysis Reagents Protease Inhibitors Phosphatase Inhibitors Detergents

Cells and Tissue

Bodily Fluids

Cell Lysis and Protein Extraction

High Abundant Protein Removal

Thermo Scientific Pierce Protein Concentrators Phosphoprotein Enrichment Kit Glycoprotein Isolation Kits Ubiquitin Isolation Kits Organelle Enrichment

Pre-fractionation & Enrichment

Thermo Scientific Slide-A-Lyzer Dialysis Casettes Thermo Scientific Zeba Desalt Spin Columns Thermo Scientific Pierce SDS-PAGE Sample Prep Kit

Sample Cleanup

2D Sample Prep Kits Thermo Scientific Imperial Stain Silver Stain for MS

Gel Separation

IgG/Albumin Removal Kits Protein A & G

In-Solution Digestion

In-Solution Tryptic Digestion Kit

In-Gel Digestion Peptide Enrichment

Phosphopeptide Isolation Kit Thermo Scientific IMAC Resin Peptide Cleanup

Mass Spectrometry

Thermo Scientific Pierce C-18 Spin Columns Thermo Scientific SCX and WCX Ion Exchange Columns Reagents: Formic Acid, TFA Instruments: Thermo Scientific LTQ Orbitrap, TSQ Quantum and LTQ FT Mass Spectrometers

Thermo Scientific LTQ Orbitrap XL Mass Spectrometer

Thermo Scientific Mass Spec Sample Prep Products. Proper sample prep means better results.

2

For more information, or to download product instructions, visit www.thermo.com/pierce

Cells and Tissue Thermo Scientific SILAC Reagents

SILAC Applications: • Quantitative analysis of relative changes in protein abundance from different cell treatments • Quantitative analysis of proteins for which there are no antibodies available • Protein expression profiling of normal vs. disease cells • Identification and quantification of hundreds to thousands of proteins in a single experiment Highlights: • Efficient – 100% label incorporation into proteins of living cells • Reproducible – eliminates intra-experimental variability caused by differential sample preparation • Flexible – media deficient in both L-lysine and L-arginine, allowing for more complete proteome coverage through dual amino acid isotope labeling • Compatible – label proteins expressed in a wide variety of mammalian cell lines adapted to grow in DMEM or RPMI 1640 medium, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3, Jurkat and others SILAC requires growing mammalian cells in specialized media supplemented with light or heavy forms of essential amino acids; i.e., 12C6 and 13C6 L-lysine, respectively. A typical experiment involves growing one cell population in medium containing light amino acids (control), while the other population is grown in the presence of heavy amino acids (experimental). The heavy and light amino acids are incorporated into proteins through natural cellular protein synthesis. After alteration of the proteome in one sample through chemical treatment or genetic manipulation, equal amounts of protein from both cell populations are then combined, separated by SDS-polyacrylamide gel electrophoresis and digested with trypsin before MS analysis. Because peptides labeled with heavy and light amino acids are chemically identical, they co-elute during reverse-phase column prefractionation and, therefore, are detected simultaneously during MS analysis. The relative peak intensities of multiple isotopically distinct peptides from each protein are then used to determine the average change in protein abundance in the treated sample (Figure 1).

Cells Grown in Light Isotope-containing Media

Cells Grown in Heavy Isotope-containing Media + Treatment

Harvest & Lyse Cells

Quantitate Extracted Protein Mix Lysates

Excise Bands

Trypsin Digestion

LC-MS/MS

SDS-PAGE

Relative Intensity

Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. The SILAC method uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry (MS) analysis for accelerated comprehensive identification, characterization and quantitation of proteins.

Ratio Determination

m/z Light

Heavy

Figure 1. Schematic of SILAC workflow. A549 cells adapted to DMEM were grown for six passages (10 days) using Thermo Scientific SILAC DMEM (Product # 89983) containing 0.1 mg/ml heavy 13C6 L-lysine-2HCl or light L-lysineHCl supplemented with 10% dialyzed FBS. After 100% label incorporation, 13C6 Llysine-labeled cells were treated with 5 µM camptothecin (Sigma, St. Louis, Product # C9911) for 24 hours. Cells from each sample (light and heavy) were lysed using Thermo Scientific M-PER Mammalian Protein Extraction Reagent (Product # 78501). Samples were normalized for protein concentration using the Thermo Scientific Pierce BCA Protein Assay (Product # 23225), and 50 mg of each sample were equally mixed before 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Gels were stained with Thermo Scientific GelCode Blue Stain Reagent (Product # 24592) and proteins were digested and alkylated using the Thermo Scientific In-Gel Tryptic Digestion Kit (Product # 89871) before analysis using an LTQ Orbitrap® Hybrid Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA).

Using a Thermo Scientific SILAC Quantitation Kit, A549 cells adapted to grow in Dulbecco’s Modified Eagle Medium (DMEM) were labeled with 13C6 L-lysine to > 98% isotope incorporation. Heavylabeled cells treated with camptothecin were lysed, mixed with control lysates, separated by SDS-PAGE and digested with trypsin before MS analysis. More than 350 proteins were successfully identified by MS/MS sequencing using a Thermo Scientific LTQ Orbitrap Mass Spectrometer. Identified peptides were then quantitated using the Thermo Scientific Bioworks Software Suite to generate SILAC ratios corresponding to relative changes in protein abundance (Figure 1).

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

3

Cells and Tissue Thermo Scientific SILAC Reagents (cont.) Most of the proteins identified had no change in abundance level after camptothecin treatment; however, 20% of proteins quantified in heavy-labeled cells had protein levels (SILAC ratios) 1.5-fold higher than control cells. One protein that was identified as being up-regulated in response to camptothecin treatment was proliferating cell nuclear antigen (PCNA), a protein with involvement in DNA repair (Figure 2). To validate SILAC data, protein levels were separately quantitated by Western blot (Figure 3). PCNA protein levels increased 1.9-fold; however, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein did not significantly change. The abundance ratios determined by Western blot were comparable to those determined by SILAC. Thermo Scientific Pierce SILAC Protein Quantitation Kits allow for complete isotopic labeling of the proteome for analysis of relative protein abundance by mass spectrometry.

Relative Abundance

Ordering Information Product # Description 89982

89983

1041.017 1041.518

100 90 80

SILAC Ratio H:L = 2.1

70 1038.508 1038.007

60 40

1039.010

30 20

1043.023

10 0 1037

1038

1039

1040

Light (L)

1041 1042 m/z Heavy (H)

1043

1044

1045

Figure 2. Representative MS spectra generated using SILAC. Light and heavy (13C6) L-lysine-containing peptides (AEDNADTLALVFEAPNQEK) from PCNA were analyzed by MS. Mass spectra of heavy peptides containing 13C6 L-lysine have an increased mass of 6 Da and are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides.

α - p53

5.9

α - β-actin α - GAPDH Light (L) Heavy (H) +

1.2

Figure 3. Comparison of A549 protein levels detected by Western blotting after camptothecin treatment. Ten micrograms of each light (L) and heavy (H) sample (Figure 1) were analyzed by 4-20% SDS-PAGE and Western blotting using specific antibodies.

SILAC Protein Quantitation Kit – DMEM

Kit

Includes: SILAC DMEM Media Dialyzed FBS 13 C6 L-Lysine-2HCl L-Lysine-2HCl L-Arginine-HCl

2 x 500 ml 2 x 50 ml 50 mg 50 mg 2 x 50 mg

500 ml

89986

Dialyzed FBS

50 ml

89987

L-Lysine-2HCl

50 mg

89988

13

50 mg

89989

L-Arginine-HCl

50 mg

89990

13

50 mg

C6 L-Lysine-2HCl 15

C6 N4 L-Arginine-HCl

Complementary Products 78430

Halt Protease Inhibitor Single-Use Cocktail (100X)

1 ea.

Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate. Protease inhibitors prepared in DMSO solution. 24 x 100 µl microtubes 0.5 M EDTA Solution (100X), 2.5 ml

Halt Protease Inhibitor Single-Use Cocktail, EDTA-free (100X)

1 ea.

Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate. Protease inhibitors prepared in DMSO solution. 24 x 100 µl microtubes

78420

Halt Phosphatase Inhibitor Cocktail

78501

M-PER® Mammalian Protein Extraction Reagent 250 ml

89826

Mem-PER® Membrane Protein Extraction Reagent

Kit

78833

NE-PER® Nuclear and Cytoplasmic Extraction Reagents

Kit

89874

Mitochondria Isolation Kit

Kit

89839

Lysosome Enrichment Kit or Tissue and Cultured Cells

Kit

89840

Peroxisome Enrichment Kit for Tissue

Kit

89841

Nuclei Enrichment Kit for Tissue

Kit

1.1

5 µM camptothecin

2 x 500 ml 2 x 50 ml 50 mg 50 mg 2 x 50 mg

SILAC DMEM Media (DMEM Medium minus L-Lysine and L-Arginine)

78425 1.9

Includes: SILAC RPMI Media Dialyzed FBS 13 C6 L-Lysine-2HCl L-Lysine-2HCl L-Arginine-HCl

89985

H:L α - PCNA

Kit

SILAC RPMI Media 500 ml (RPMI-1640 Medium minus L-Lysine and L-Arginine)

1042.521

1039.512

Pkg. Size

SILAC Protein Quantitation Kit – RPMI 1640

89984

1042.020

50

1036

References: 1. Everly, P.A., et al. (2004). Quantitative cancer proteomics: Stable isotope labeling with amino acids (SILAC) as a tool for prostate cancer research. Mol & Cell Proteomics. 3.7: 729-735. 2. Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nature Reviews. 7: 952-959. 3. Levine, A.J. (1997). p53, the cellular gatekeeper for growth and division. Cell. 88: 323–331.

1 ml

The purchase of this product conveys a non-transferable license to the Purchaser to use this product in methods protected under U.S Patent 6,653,076 (owned by University of Washington) for research purposes only.

4

For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Cell Surface Protein Isolation Kit

Adherent or suspended cells are first incubated with Sulfo-NHS-SSBiotin, a cleavable reagent. The cells are lysed with a mild detergent and labeled proteins are isolated with immobilized NeutrAvidin Resin. The bound proteins are recovered by incubating the resin with SDS-PAGE sample buffer containing 50 mM DTT. The reducing agent cleaves the disulfide bond within the spacer arm of the biotinylation reagent (Figure 5). Nearly 100% of the bound proteins are released (Figure 4). The protocol is optimized for diverse cell lines including NIH 3T3, HeLa, C6 and A431. Isolated proteins can be analyzed by Western blot, allowing for differential expression analysis between treated and untreated cells (Figure 6) or between two or more cell lines.

Convenient biotinylation and isolation of cell surface proteins for Western blot analysis. Highlights: • Isolates cell surface proteins – reduces complexity of lysates • Efficiently recovers labeled proteins – cleavable biotin allows for nearly 100% recovery of isolated cell surface proteins • Convenience – includes all reagents and complete instructions for labeling, lysis and purification • Western blotting applications – proteins recovered in SDS-PAGE buffer are loaded directly onto polyacrylamide gels • Robust system – protocol designed for diverse cell lines

EGF

The Thermo Scientific Cell Surface Protein Isolation Kit specifically targets mammalian cell surface proteins to the exclusion of intracellular proteins. The kit efficiently labels proteins with accessible lysine residues and sufficient extracellular exposure (Figure 4).

-

+

-

+

EGF

Integrin α5

-

-

+

+

EGFR

-

+

-

+

Integrin β1

A. A431

B. HeLa

Figure 6. Differential expression of cell surface proteins in response to EGF. A431 and HeLa cells were treated with or without 20 ng/ml and 10 ng/ml EGF for 16 hours, respectively. Both cell types were processed with the Thermo Scientific Cell Surface Protein Isolation Kit protocol. Elution fractions were analyzed by Western blot for the quantities of A. integrin β1 and integrin α5 subunits or B. EGFR. Thermo Scientific GelCode Blue Safe Stain

Supplier I

Ordering Information Product # Description 89881

Supplier B

Pkg. Size

Cell Surface Protein Isolation Kit

8 applications

Includes: EZ-Link™ Sulfo-NHS-SS-Biotin Quenching Solution Lysis Buffer Immobilized NeutrAvidin Gel

8 x 12 mg vials 16 ml 4.5 ml 2.25 ml settled gel supplied as 50% slurry (4.5 ml total volume) 34 ml 8 spin columns with caps and collection tubes

Supplier S

Wash Buffer Column Accessory Pack

Figure 4. Specificity of isolation. HeLa cells were treated with or without Sulfo-NHS-SS-Biotin and processed with the Thermo Scientific Cell Surface Protein Isolation Kit protocol. Elution fractions, post-elution resin and flow-through were analyzed by Western blot for A. cell surface proteins EGFR, IGF-1Rβ, integrin β1 and integrin α5 and B. intracellular proteins, including heat shock protein 90™ (hsp90) and calnexin. Legend: (+) label, (-) no label, (F) flow-through, (R) Thermo Scientific NeutrAvidin™ Gel and (E) elution. Only labeled cell surface proteins are present in the elution fractions. Quench Reaction

Biotinylate cells

No-Weigh™ Dithiothreitol (DTT) BupH Phosphate Buffered Saline BupH Tris Buffered Saline

Transfer cell pellet to 1.5 ml tube 45 40

8 x 7.7 mg microtubes 2 packs 1 pack

Isolate biotinylated proteins on Thermo Scientific NeutrAvidin Gel

35 30 25

Harvest Cells

30 minutes at 4˚C

20 15 10 75 50

N gel

N

N B

Wash gel then elute with SDS-PAGE sample buffer + 50 mM DTT

gel

Lyse cells 30 minutes on ice 1-D gel

N +

protein – SH

Perform electrophoresis or other application

B SH

S-S-protein

N

Thermo Scientific NeutrAvidin Biotin-Binding Protein

B

Biotin

Figure 5. Procedure for the Thermo Scientific Cell Surface Protein Isolation Kit.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

5

Thermo Scientific Products for Cell Lysis and Protein Extraction Table 1. Thermo Scientific Cell Lysis Reagents selection guide. Dialyze1 Compatibility

Description

Organisms/Samples

B-PER® Reagent† 78243, 165 ml 78248, 500 ml

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains BL21(D3)> JM109> DH5a >M15, Archaebacteria, nematodes and Acinetobacter sp.

Yes

Reporter assays, IPs2, Western blot, GST- and histidine-tag purification

B-PER II Reagent 78260, 250 ml (A 2X version of B-PER Reagent)

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains BL21(D3)> JM109> DH5α>M15, Archaebacteria, nematodes and Acinetobacter sp.

Yes

Reporter assays, IPs2, Western blot, GST- and histidine-tag purification

B-PER PBS Reagent 78266, 500 ml

Gram(-) bacteria, S. aureus, H. pylori, E. coli strains BL21(D3)> JM109> DH5α>M15, Archaebacteria, nematodes and Acinetobacter sp.

Yes

Reporter assays, IPs2, Western blot, GST- and histidine-tag purification

Y-PER® Reagent 78991, 200 ml 78990, 500 ml

S. cerevisiae, Schizo-saccharomyces pombe, C. albicans, B. subtilis, E. coli, P. pastoris, Strep. avidinii and Acinetobacter sp.

No

IPs2, Western blot, β-Gal enzyme assays, IEF after dialysis, GST- and histidine-tag purification

Y-PER Plus Reagent 78998, 25 ml 78999, 500 ml

Yeast (S. cerevisiae) and Acinetobacter sp.

Yes

GST- and histidine-tag purification, Western blot

M-PER Reagent 78503, 25 ml 78501, 250 ml 78505, 1 L

Cultured mammalian cells, COS-7, NIH 3T3, Hepa 1-6, 293, CHO, MDA, MB231 and FM2

Yes

Luciferase, β-Gal (low signal), CAT, kinase assays, ELISAs, immobilized glutathione, Western blot

P-PER® Plant Protein Extraction Reagent 89803, Kit

Multiple plant organs (leaf, stem, root, seed and flowers); multiple plant species (Arabidopsis, tobacco, maize, soybeans, peas, spinach, rice and other plant tissues); and fresh, frozen and dehydrated plant tissues

No

1-D and 2-D gel electrophoresis, Western blotting, activity assays and protein affinity purifications*

T-PER® Reagent 78510, 500 ml

Heart, liver, kidney and brain

Yes

Luciferase, β-Gal, CAT, kinase assays, Western blot, ELISAs, immobilized glutathione

I-PER® Reagent 89802, 250 ml

Baculovirus-infected insect cells grown in suspension or monolayer culture

No

Western blot, 6xHis-tagged protein purification, protein assays and ion-exchange chromatography

NE-PER Reagent 78833

Tissue: calf liver. Cultured cells: epithelial (HeLa), fibroid (COS-7), kidney (NIH 3T3), liver (Hepa 1) and brain (C6)

Mem-PER Reagent 89826

Cultured cells: brain (C6), epithelial (HeLa), fibroblasts (NIH 3T3) and yeast (S. cerevisiae)

Yes4

Western blot and 2-D6

Mitochondria Isolation Kit for Cultured Cells† 89874 Mitochondria Isolation Kit for Tissue† 89801

Mammalian cells

Yes7

Western blot, 2-D Western blots, electrophoresis. Applications include apoptosis, signal transduction and metabolic studies.

Pierce RIPA Buffer 89900, 100 ml 89901, 250 ml

Cultured mammalian cells and cytoplasmic, membrane and nuclear proteins

Yes

Reporter assays, protein assays, immunoassays and protein purification

Lysosome Enrichment Kit for Tissues and Cultured Cells 89839

Tissues and Cultured Cells

N/A

2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies

Peroxisome Enrichment Kit for Tissue 89840

Heart, liver, kidney and brain

N/A

2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies

Nuclei Enrichment Kit for Tissue 89841

Heart, liver, kidney and brain

N/A

2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies

No (CER) EMSA (if using < 3 µl or 10%, otherwise dialyze first in Yes (NER) SAL MINIs5), Western blot, reporter assays, IEF (after dialysis to reduce salt concentration) and 2-D6

Heart, liver, kidney and brain

1. The detergent can be removed by dialysis 2. Immunoprecipitation 3. Halt Protease Inhibitor Cocktail, Product # 78410 and 78415 (EDTA-free) 4. Samples prepared in Mem-PER Reagent can be dialyzed if the buffer contains detergent (e.g., CHAPS), otherwise use Thermo Scientific Advanced Sample Clean-Up Kit (Product # 89888) 5. Slide-A-Lyzer MINI Dialysis Units 6. 2-D Sample Prep for Nuclear Proteins (Product # 89863) and 2-D Sample Prep for Membrane Proteins (Product # 89864) are designed using our popular NE-PER and Mem-PER Reagents. 7. Need to lyse mitochondria first. *Although kit works without liquid nitrogen/freeze-grinding, Dounce homogenization, blade-shearing or glass-bead agitation for cell disruption, it is compatible with these alternative mechanical aids. † See patent information.

6

For more information, or to download product instructions, visit www.thermo.com/pierce

Protein Assay Compatibility

Notes

Pierce BCA Assay and Coomassie Plus Assay

Protease inhibitors3 may be added to prevent protein degradation. Salts, chelating agents and reducing agents can be added for more efficient lysis. Do not exceed 0.5 M NaCl. Better lysis if cells are frozen in B-PER Reagent.

Pierce BCA Assay and Coomassie Plus Assay after Compat-Able™ Protein Assay Reagent Set (Product # 23215) or dilute two to four times

Protease inhibitors3 may be added to prevent protein degradation. Salts, chelating agents and reducing agents can be added for more efficient lysis. Better lysis if cells are frozen in B-PER Reagent.

Pierce BCA Assay and Coomassie Plus Assay after Compat-Able Protein Assay Reagent Set (Product # 23215) or dilute two to four times

Protease inhibitors3 may be added to prevent protein degradation. Salts, chelating agents and reducing agents can be added for more efficient lysis. Better lysis if cells are frozen in B-PER Reagent.

Pierce BCA Assay

Protease inhibitors3 may be added to prevent protein degradation. Use at room temperature. Double incubation time for use at 4°C. Use log-phase cells. For stationary phase cells, add 0.1 M DTT or 20-50 mM TCEP. Will work with 1 mM EDTA. Does not lyse spores. Cannot use with ion exchange columns.

Pierce BCA Assay and Coomassie Plus Assay

Protease inhibitors3 may be added to prevent protein degradation. The addition of up to 2 M NaCl may result in increased efficiency of lysis and protein yield.

Pierce BCA Assay and Coomassie Plus Assay

Protease inhibitors3 may be added to prevent protein degradation. Adding 150 mM NaCl results in increased efficiency of lysis and higher protein yield in some cells lines. A PBS rinse of cells prior to lysis removes contaminants such as phenol red and increases protein yield.

Pierce BCA Assay, Reducing Agent-Compatible Not compatible with Bradford, Coomassie or Pierce BCA Assay

Kit lyses most plant cells without harsh mechanical lysis aids; extremely fibrous tissues such as woody stems may require mechanical grinding by devices not included in this kit. P-PER Extracts can be quantified using the Pierce BCA Protein Assay Kit, Reducing Agent Compatible (Product # 23250).

Pierce BCA Assay (dilute 1:1) and Coomassie Plus Assay

Protease inhibitors3 may be added to prevent protein degradation. Mechanical disruption of the tissue is still required. Can also be used for cultured cells.

Pierce BCA Assay

Protease inhibitors3 may be added to prevent protein degradation.

Pierce BCA Assay and Coomassie Plus Assay (dilute CER Reagent mixture four times)

Protease inhibitors3 may be added to prevent protein degradation. Packed cell vol.: 2 x 106 HeLa cells = 10 µl = 20 mg. Tissue yield (calf liver): 3-4 mg cytoplasmic protein/100 mg tissue; 1-1.5 mg nuclear protein/100 mg tissue. Cell yield (HeLa): 300-400 µg cytoplasmic protein/106 cells; 40-60 µg nuclear protein/106 cells. Positive controls tested: cytoplasmic (β-Gal, PKC, Hsp90); nuclear (Oct-1, p53, DNA polymerase).

Pierce BCA Assay and Coomassie Plus Assay; hydrophobic phase needs to be dialyzed first; see instruction book

Protease inhibitors3 may be added to prevent protein degradation. Can dialyze against another detergent (e.g., CHAPS). Extraction efficiency is generally > 50% with the cell lines tested (having proteins with up to two transmembrane segments).

Pierce BCA Assay (after lysis)

Protease inhibitors may be added to prevent protein degradation. Douncing will increase isolation efficiency vs. detergent alone; however, multiple samples can be processed simultaneously using the reagent-based methods.

Pierce BCA Assay

Protease inhibitors3 may be added to prevent proteolysis and maintain phosphorylation of proteins

Coomassie Plus – The Better Bradford Assay Kit

Protease inhibitors3 may be added to prevent proteolysis and maintain phosphorylation of proteins

Coomassie Plus – The Better Bradford Assay Kit

Protease inhibitors3 may be added to prevent proteolysis and maintain phosphorylation of proteins

Coomassie Plus – The Better Bradford Assay Kit

Protease inhibitors3 may be added to prevent proteolysis and maintain phosphorylation of proteins

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

7

Thermo Scientific Products for Cell Lysis and Protein Extraction Halt Protease Inhibitor Cocktails

0%

New single-use, ready-to-use protease inhibitor cocktails. Proteases make up a large category of enzymes, including endopeptidases and exopeptidases, which catalyze the hydrolytic breakdown of proteins into peptides or amino acids in a wide variety of cell and tissue types. This breakdown can lead to the loss of a large number of valuable proteins, adversely affecting downstream applications. Thermo Scientific Halt Single-Use Protease Inhibitor Cocktails help preserve the integrity of target proteins intended for downstream analysis. These cocktails target all major classes of proteases including aspartic acid, cysteine, serine and metalloproteases. AEBSF, aprotinin, bestatin, E64, leupeptin and pepstatin A, all known potent inhibitors of proteases, have been expertly formulated in a ready-to-use cocktail and specially packaged to maintain product integrity and offer a new level of convenience. Ready-to-use Halt Protease Inhibitor Cocktails are conveniently packaged in single-use microtubes, each of which contain sufficient reagent to treat up to 10 ml of sample. Our Protease Inhibitor Cocktails are the convenient alternative to protease inhibitor tablets, saving time, eliminating the mess of splitting tablets and providing a measure of consistency because you do not have to use a razor blade to treat samples that are < 10 ml (Figure 1). Each Halt Single-Use Cocktail contains 24 x 100 µl sealed microtubes sufficient to treat up to 240 ml of cell extract. As a 100X concentrate in DMSO, the single-use format provides access to only the cocktail needed to knock-down protease activity in up to 10 ml of cell lysate per microtube. The Halt Single-Use Cocktail contains reversible and irreversible inhibitors for serine-, cysteine-, aspartic acid-proteases and aminopeptidases present in virtually all cellular lysate samples. A 0.5 M EDTA solution is provided with one formulation to promote the inhibition of metalloproteases.

59%

61%

97%

98% Control; Extract Only

Protease Inhibitor Thermo Scientific Halt Cocktail Tablet Single-Use Cocktail

Tablet; EDTA-Free

Thermo Scientific Halt Single-Use EDTA-Free Cocktail

Percent Inhibition

Figure 1. Thermo Scientific Halt Protease Inhibitor Single-use Cocktails are more effective than tablet-format cocktails. Using a validated protease assay and 1.0 mg/ml of rat pancreas extract, the Halt Protease Inhibitor Single-Use Cocktails, with and without EDTA added, were tested against commercially available tablet-format protease inhibitor cocktails under the same conditions. A 1X final concentration of each inhibitor was added. The single-use formulation resulted in ≥ 97% inhibition compared to ≥ 59% inhibition for the tablet.

Each microtube in the strip is heat-sealed with a foil that can be easily punctured by a pipette tip. Only that amount of protease inhibitor cocktail needed for the application is removed from the microtube. The remaining tubes in the strip are preserved and protected from exposure to air, moisture and other potential contamination until needed for subsequent applications. Although many manufacturers won’t disclose the composition or concentration of their protease inhibitors, we publish the exact identity and concentration of each cocktail component (Table 2).

Table 2. Thermo Scientific Halt Protease Inhibitor Single-Use Cocktail formulation and concentrations.

8

Protease Inhibitor

MW

Protease Family Targeted

Inhibitor Type

Solubility (Solvent system)

Concentration of Each Inhibitor in the Protease Inhibitor Cocktails (100X in DMSO) (Product # 78430 and 78425)

AEBSF•HCl

239.5

Serine proteases

Irreversible

200 mg/ml (H2O)

100 mM

Aprotinin

6511.5

Serine proteases

Reversible

10 mg/ml (H2O)

80 µM

Bestatin

308.38

Amino-peptidases

Reversible

5 mg/ml (methanol)

5 mM

E-64

357.4

Cysteine proteases

Irreversible

20 mg/ml (1:1 EtOH/H2O)

1.5 mM

EDTA (not included in Product # 78425)

372.24

Metalloproteases (Chelates divalent cations)

Reversible

10 g/100 ml (H2O)

0.5 M

Leupeptin

475.6

Serine and cysteine proteases

Reversible

1 mg/ml (H2O)

2 mM

Pepstatin A

685.9

Aspartic acid proteases

Reversible

1 mg/ml (MeOH)

1 mM

For more information, or to download product instructions, visit www.thermo.com/pierce

Highlights: • Single-use, ready-to-use packaging keeps protease inhibitors fresh, reduces the risk of reagent contamination and minimizes waste • Innovative microtube design allows immediate inhibition of lysates, no thawing or waiting for tablets to dissolve • EDTA-containing and EDTA-free formulations published – know exactly what is in the cocktail • Compatible with Thermo Scientific Pierce Cell Lysis Buffers (Figure 2) • Effective for suppressing proteolytic activity in detergent-based cell lysis reagents • Outstanding performance demonstrated with Thermo Scientific M-PER Mammalian, B-PER Bacterial, T-PER Tissue and Y-PER Yeast Protein Extraction Reagents • Stable for one year when refrigerated • Same high-quality protease inhibitor components available separately; prepare a custom cocktail or increase the concentration of a class of protease inhibitor in the formulation to improve the stability or recovery of a specific target protein 1

2

3

4

1

2

3

Protease Inhibitor References 1. Kulakowska-Bodzon, A., et al. (2006). Methods for samples preparation in proteomic research. J. Chromatogra. B. (doi:10.1016/j.jchromb.2006.10.040). 2. www.mnhn.fr/mnhn/bpy/2DMS/2DE.pdf: PART I Sample Preparation, p.9 (2.2 Protection against proteolysis) 3. North, M.J. (1989). Prevention of unwanted proteolysis in Proteolytic Enzymes: A Practical Approach (Beynon, R.J., Bond, J.S. eds), pp. 105-124, IRL Press, Oxford. 4. Salvensen, G. and Nagase, H. (1989). Inhibition of proteolytic enzymes in Proteolytic Enzymes: A Practical Approach (Beynon, R.J., Bond, J.S. eds). pp. 83-104, IRL Press, Oxford.

Ordering Information Product # Description 78430

Halt Protease Inhibitor Single-Use Cocktail (100X)

Pkg. Size 1 ea.

Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate. Protease inhibitors prepared in DMSO solution. 24 x 100 µl microtubes 0.5 M EDTA Solution (100X), 2.5 ml

78425

Halt Protease Inhibitor Single-Use Cocktail, EDTA-free (100X)

1 ea.

Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate. Protease inhibitors prepared in DMSO solution. 24 x 100 µl microtubes

4

Also available in new sizes: 78429

Halt Protease Inhibitor Cocktail

5 ml

78437

Halt Protease Inhibitor Cocktail, EDTA-free

5 ml

78438

Halt Protease Inhibitor Cocktail

10 ml

78439

Halt Protease Inhibitor Cocktail, EDTA-free

10 ml

Individually Packaged Protease Inhibitors 78431

AEBSF

100 mg

[4-(2-aminoethyl)benzenesulfonyl fluoride•HCl]

78432

Aprotinin

25 mg

78433

Bestatin

10 mg

N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl]-L-leucine

78434

A. Thermo Scientific B-PER Bacterial Protein Extraction Reagent

B. Thermo Scientific M-PER Mammalian Protein Extraction Reagent

E-64

10 mg

[N-trans-epoxysuccinyl-L-leucine-4 guanidinobutylamide] Also known as N-[N-(L-3-trans-carboxirane-2-carbonyl)] -L-leucyl agmatine

78435

Leupeptin

50 mg

(acetyl-leucyl-leucyl-argininal)•Hemisulfate

Figure 2. Thermo Scientific Halt Protease Inhibitor Single-Use Cocktails are compatible with Thermo Scientific M-PER Mammalian Protein Extraction and B-PER Bacterial Protein Extraction Reagents. Bovine serum albumin (BSA) was incubated overnight at 37°C in the presence of 0.1 mg/ml trypsin in M-PER Extraction Reagent and B-PER Extraction Reagent, respectively. Lane 1. Control 1, BSA only; Lane 2. Control 2, Trypsin (0.1 mg/ml) (+) BSA and no Halt Protease Inhibitor Single-Use Cocktail present; Lane 3. BSA (+) Trypsin with Halt Protease Inhibitor Single-Use Cocktail with EDTA; and Lane 4. BSA (+) Trypsin with Halt Protease Inhibitor Single-Use Cocktail. Under extreme time and temperature conditions, substantial protection from degradation was observed as marginal to barely detectable cleavage products of BSA.

78436

Pepstatin A

25 mg

Compatible Products 78420

Halt Phosphatase Inhibitor Cocktail

1 ml

1 ml of cocktail protects up to 100 ml of sample. A mixture of four phosphate inhibitors including sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate.

89806

Protein Stabilizing Cocktail

10 ml

4X Concentrated Solution

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

9

Thermo Scientific Products for Cell Lysis and Protein Extraction Halt Phosphatase Inhibitor Cocktails

MEK –

PMEK +

–

+

–

+

–

+

Offering protection from both serine/threonine and protein tyrosine phosphatases. Highlights: • All-in-one format – inhibits both serine/threonine and protein tyrosine phosphatases • Compatible – works with standard protein assays, including Pierce BCA Protein Assay Kit and Coomassie Plus (Bradford) Assay • User-friendly storage at 2-8˚C – saves valuable space in the freezer • Convenient size – 1 ml of cocktail protects up to 100 ml of sample Phosphorylation and dephosphorylation is a molecular on/off switch that regulates many key biological pathways within the cell, including signal transduction, cell division and apoptosis. The Thermo Scientific Halt Phosphatase Inhibitor Cocktail (Product # 78420) preserves phosphorylation of proteins in cells and tissues. The cocktail contains a mixture of four phosphatase inhibitors of broad specificity, including sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate. Unlike other commercially available phosphatase inhibitor cocktails that protect against either serine/threonine phosphatases or protein tyrosine phosphatases, Halt Phosphatase Inhibitor Cocktail protects phosphoproteins from both families of phosphatases (Figure 3). Conveniently packaged in 100X format, the cocktail is stable at 4˚C and is compatible with common protein assays, including the BCA Protein Assay Kit (Product # 23225) and Bradford-based assays such as the Coomassie Plus (Bradford) Assay (Product # 23236).

A. MAPK –

PMAPK +

B. STAT3 –

PSTAT3 +

C.

Figure 3. Thermo Scientific Halt Phosphatase Inhibitor Cocktail preserves phosphorylation of MEK, MAP Kinase 42/44 and STAT3 in HeLa cell lysate. Cells were lysed in the absence (-) and presence (+) of Halt Phosphatase Inhibitor Cocktail. Lysates were analyzed by Western blot for total and phosphorylated protein as indicated. A. MEK and phosphorylated MEK (PMEK), B. MAP kinase and PMAP kinase and C. STAT3 and PSTAT3. The proteins are phosphorylated on serine, threonine/tyrosine and tyrosine, respectively.

Ordering Information Product # Description 78420

Halt Phosphatase Inhibitor Cocktail

Pkg. Size 1 ml

Sufficient reagent to protect up to 100 ml of sample.

78426

Halt Phosphatase Inhibitor Cocktail

5 x 1 ml

78427

Halt Phosphatase Inhibitor Cocktail

10 ml

78428

Halt Phosphatase Inhibitor Single-Use Cocktail

24x 100 µl

Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate.

10

For more information, or to download product instructions, visit www.thermo.com/pierce

Protein Stabilizing Cocktail Extends shelf-life of precious proteins. Highlights: • Stabilizes proteins more than buffer alone • Does not destabilize biomolecules in downstream assays • All components are dialyzable • Easy to pipette (vs. 50% glycerol) Protein Classes Tested: • Kinase • Peroxidase • Cytokine • Restriction Enzyme • Phosphatase • Luciferase • Antibody

Although the degree of stabilization is protein-specific, the cocktail significantly stabilizes proteins better than conventional buffer alone. The Protein Stabilizing Cocktail is nontoxic. If needed, all cocktail components can be removed by dialysis or desalting before use in downstream assays. Visit our website to view data, including luciferase activity stabilized by the Protein Stabilizing Cocktail.

Ordering Information Product # Description 89806

Protein Stabilizing Cocktail, 4X Concentrated Solution

Pkg. Size 10 ml

Sufficient reagent to make 40 ml of storage solution.

The Thermo Scientific Protein Stabilizing Cocktail is a versatile solution that increases the shelf-life of purified or partially purified proteins during routine storage. The proprietary formulation of low-molecular weight, naturally occurring molecules helps protect proteins from environmental stresses that can otherwise lead to enzyme inactivation, aggregation and freeze-thaw damage.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

11

Thermo Scientific Products for High Abundant Proteins SwellGel Blue Albumin Removal Kit Offers ease-of-use, speed and performance not found in other kits. Thermo Scientific SwellGel Blue Albumin Removal Discs are for high-capacity albumin removal from small (10-100 µl) serum samples. Each disc can bind approximately 2 mg of human serum albumin.* Just add water and the discs hydrate in less than 20 seconds to form an equilibrated chromatographic resin bed of approximately 200 µl. Processed cells are ready for immediate downstream analysis by 2-D or MS applications. The simplicity of sample preparation and the versatility offered by the SwellGel Technology make it ideal for standard chromatography, 2-D and micro-spin column applications. Albumin Contamination

Albumin Depleted

Highlights: • Excellent for 2-D applications – allows you to study less abundant proteins without interference from albumin (Figure 1) or processing buffers • Convenient and versatile – kit includes everything needed to remove albumin, also works with 96-well filter plates • Room temperature storage – discs do not leak or spill, and the kit stores conveniently on bench top • Pre-measured, easy-to-handle discs with an easy-to-follow protocol (Figure 2) – no waste – all resin can be used; no need to pipette messy slurries SwellGel Blue Albumin Removal Kits contain ready-to-hydrate Cibacron™ Blue Activated Discs, binding/wash buffer and Mini-Spin Columns. Separations are performed in microcentrifuge columns and can be processed by low-speed centrifugation or syringe-based methods. Reference 1. Steel, L.F., et al. (2003). Efficient and specific removal of albumin from human serum samples. Mol Cell Proteomics 2, 262-270.

Ordering Information A.

B.

Product # Description Figure 1. Albumin removal for 2-D analysis. Serum sample obtained by diluting 10 µl human serum with 40 µl TBS and loading 5 µl onto Gel A. Albumin-free sample obtained by diluting 50 µl human serum 1:1 with buffer, adding it to a Thermo Scientific SwellGel Blue Disc, washing the resin three times with 50 µl of buffer, and combining the fractions; loading 5 µl onto Gel B. Both samples were focused using pH 4-7 IEF strips and run on 8-16% Tris-glycine gels.

Step 1. Hydrate Thermo Scientific Disc in water. Spin.

SERUM

FILTRATE

Step 2. Add serum sample to the resin. Incubate 2 minutes.

Step 3. Recover filtrate and add to the resin. Incubate 2 minutes and spin.

89845

SwellGel Blue Albumin Removal Kit* Includes: SwellGel Blue Albumin Removal Discs Binding/Wash Buffer Mini-Spin Columns

Step 5. Combine desired fractions.

Figure 2. Thermo Scientific SwellGel Blue Albumin Removal Kit Protocol.

12

12-25 Reactions 25 discs 6.25 ml 25 columns

*SwellGel Blue Discs have differing levels of affinity for species-specific albumins. SwellGel Blue Discs bind human, swine and sheep albumin. They can also be used with a minor protocol change for bovine, calf and goat albumin. They do not bind mouse albumin.

BUFFER

Step 4. Add Binding/Elution Buffer.

Pkg. Size

For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Albumin/IgG Removal Kits Process multiple 2-D or 2D/LC samples in less than 40 minutes. Low-abundant proteins in human serum and plasma can provide information about human diseases. However, human fluid analysis is often complicated by high concentrations of albumin and IgG, which accounts for up to 70% of total serum protein (Figure 4). We offer several kits for removing albumin and/or IgG from samples, allowing analysis of low-abundant proteins. For Detailed Proteomic Analysis The Thermo Scientific Pierce Albumin/IgG Removal Kit (Product # 89875) contains a mixed bed matrix of immobilized Cibacron Blue Dye and high-capacity immobilized Protein A and provides an efficient method for depleting excess abundant proteins while enriching the lower molecular weight proteins, peptides and other small components. The kit is formulated for depleting human serum albumin (HSA) and the major subclasses of immunoglobulin (IgG) from serum, plasma or spinal fluids. An Economical – Yet Effective – Method Thermo Scientific Pierce Antibody-Based Albumin/IgG Removal Kit (Product # 89876) contains an Immobilized Anti-IgG/Anti-Albumin Gel (immobilized antibodies are polyclonal from goat) for removing HSA and all major subclasses of gamma globulin (IgG) from human serum plasma or spinal fluids. This kit uses highly specific antibodies, providing minimal nonspecific interactions. This product can deplete greater than 95% of HSA and 90% of IgG. Each kit is optimized for downstream analysis by 2-D electrophoresis or 2D/LC mass spectrometric methods and allows for multiple samples to be processed simultaneously.

Figure 4. 2-D gel of human serum processed with the Thermo Scientific Pierce Antibody-Based Albumin/IgG Removal Kit. Approximately 30 µg of processed human serum was isoelectric-focused on a pH 5-8 strip and then separated in the second dimension on an 8-16% SDS-polyacrylamide gel. Thermo Scientific Silver Stain II (Product # 24612) was used to visualize bands. With albumin and IgG removed, low-abundant proteins are readily identified.

Ordering Information Product # Description 89875

89876

Kit

Sufficient material for up to 25 samples of 600 µg of serum. Includes: Dye/Protein A Resin (sold as 50% slurry [4.25 total volume]) Spin Columns and Accessories

2.13 ml resin bed 27

Pierce Antibody-Based Albumin/IgG Removal Kit Sufficient material for up to 12 samples of 600 µg of serum. Includes: Antibody-Based Resin (sold as 62% slurry [7.25 total volume])

Spin Columns and Caps

Albumin

Pkg. Size

Pierce Albumin/IgG Removal Kit

Kit

4.5 ml of immobilized anti-HSA/ anti-IgG resin bed 12

lgG Heavy Chain

lgG Light Chain

Figure 3. 2-D gel unprocessed human serum. Approximately 30 µg of unprocessed human serum was isoelectric-focused on a pH 5-8 strip and then separated in the second dimension on an 8-16% SDS-polyacrylamide gel. Thermo Scientific Silver Stain II (Product # 24612) was used to visualize bands. Large poorly resolved bands corresponding to human serum and IgG obscure much of the gel.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

13

Thermo Scientific Products for Pre-fractionation and Enrichment Pierce Phosphoprotein Enrichment Kits Process cell and tissue samples in less time and with greater purity. Phosphorylation is one of the most frequently occurring posttranslational modifications in proteins. It is estimated that as many as 30% of all cellular proteins are transiently phosphorylated on serine, threonine and tyrosine residues. Reversible protein phosphorylation regulates nearly all intracellular biological events, including signal transduction, protein-protein interactions, protein stability, protein localization, apoptosis and cell-cycle control. Deregulation of protein phosphorylation is a hallmark of numerous human diseases, including cancer and metabolic and immune disorders. Detecting changes in protein phosphorylation can be a difficult task because of the transient labile state of the phosphate group. Furthermore, low phosphoprotein abundance and poorly developed phospho-specific antibodies contribute to difficulties in phosphoprotein detection. Recent advances in mass spectrometry technology in combination with phosphoprotein enrichment using immobilized metal affinity chromatography (IMAC) have resulted in greater resolution of the phosphoproteome. The new Thermo Scientific Pierce Phosphoprotein Enrichment Kit efficiently enriches phosphorylated proteins derived from mammalian cells and tissues. The proprietary metal and buffer composition produces superior yields with negligible nonspecific binding.

Phospho-specific antibodies recognizing key regulatory proteins involved in growth factor signaling were used to monitor binding specificity of our Phosphoprotein Enrichment Kit (Figure 1). Specificity of the kit is further demonstrated by the absence of Cytochrome C (pI 9.6) and p15Ink4b (pI 5.5), two proteins not predicted to be phosphorylated, in the elution fraction and their emergence in the flow-through and wash fractions (Figure 1). Furthermore, dephosphorylation of HeLa cell extract in vitro resulted in diminished binding of PTEN, MAPK and GSK3β to the Pierce Phosphoprotein Enrichment Column as evidenced by their absence in the elution fraction. Conversely, all three proteins were present in the elution fraction from non-treated HeLa extract (Figure 2). Our Phosphoprotein Enrichment Kit provided superior and efficient phosphoprotein enrichment yields when compared to competitors’ products (Table 1). It also effectively enriched phosphoproteins from homogenized mouse liver tissue (Figure 3). Table 1. The Thermo Scientific Pierce Phosphoprotein Enrichment Kit provides higher phosphoprotein yields in less time than competitors’ kits. Kit Thermo Scientific Pierce Phosphoprotein Enrichment Kit

Yield (%)

Enrichment Time (Hours)

15

1.5

Supplier Q Kit

4.4

4.5

Supplier I Kit

2.6*

3.5

Supplier C Kit

8

3

Supplier E Kit

Too dilute to determine

5

* Based on maximum 1 mg load per manufacturer’s protocol.

Highlights: • Specific – low contamination from nonspecific proteins • Fast – easy-to-use spin format enriches of phosphorylated proteins in less than 2 hours • Superior yield – high yield from complex biological samples, cell culture lysate and mouse tissue extract • Convenient format – complete kit includes pre-dispensed spin columns, buffers, reagents and Thermo Scientific Pierce Protein Concentrators • Compatible – works with downstream applications, including mass spectrometry, Western blotting and 2D-PAGE

14

For more information, or to download product instructions, visit www.thermo.com/pierce

FT

W

E

L10

L

FT

W

E

L25 Phospho-PTEN S380

HeLa + EGF

Phospho-MAPK T202/Y204

Phospho-Rb S795

Phospho-PTEN S380

Cytochrome C

Phospho-GSK3β S9

Phospho-MAPK T202/Y204

NIH 3T3 + PDGF

Phospho-Akt S473

Phospho-Src Y527

Cytochrome C

p15Ink4b

Figure 1. Highly pure phosphoprotein enrichment from complex biological samples. Serum-starved HeLa and NIH 3T3 cells were stimulated with EGF and PDGF, respectively. Cell lysate (2 mg) was used for enrichment. Concentrated flow-through, wash and elution fractions were resolved by SDS-PAGE. Gel lanes were normalized by protein concentration (10 µg/lane). Western blot analysis was performed using antibodies that detect site-specific phosphorylation events. Cytochrome C (pI 9.6) and p15Ink4b (pI 5.5) served as negative controls for nonspecific binding of non-phosphorylated proteins. FT = flow-through fraction, W = pooled wash fractions, E = pooled elution fractions and L = non-enriched total cell lysate.

Total Cell Lysate 25 µg λ Pase +

10 µg +

Figure 3. Efficient enrichment of phosphoproteins from mouse liver extract. Homogenized mouse liver extract (~2 mg) was loaded onto a Thermo Scientific Pierce Phosphoprotein Enrichment Column. Concentrated flow-through, wash and elution fractions were resolved by SDS-PAGE. Gel lanes were normalized by protein concentration (10 µg/lane). Western blot analysis was performed using antibodies that detect site-specific phosphorylation events. Cytochrome C (pI 9.6) served as a negative control for nonspecific binding. L10 = non-enriched total cell extract (10 µg), FT = flow-through fraction, W = wash fraction, E = elution fraction and L25 = non-enriched total cell extract (25 µg).

Ordering Information Product # Description 90003

Pkg. Size

Pierce Phosphoprotein Enrichment Kit

Kit

Includes: Phosphoprotein Enrichment Column Resin Bed (1 ml) Lysis/Binding/Wash Buffer Elution Buffer CHAPS White Column Caps Pierce Protein Concentrator 7 ml/9K MWCO

10 ea. 325 ml 60 ml 1g 10 Devices

Elution 10 µg + Phospho-PTEN S380

Phospho-MAPK T202/Y204

Phospho-GSK3β S9

Figure 2. Highly specific phosphoprotein purification from lambda phosphatase-treated cells. Non-treated and lambda dephosphorylated HeLa cell extract (2 mg) was loaded onto separate Thermo Scientific Pierce Phosphoprotein Enrichment Columns. Concentrated elution fractions were resolved by SDS-PAGE. Gel lanes were normalized by protein concentration (10 µg/lane). To determine enrichment, 10 µg and 25 µg of non-treated or lambda phosphatase-treated total cell extract (non-enriched) was loaded onto each gel. Western blot analysis was performed using phospho-specific antibodies recognizing key proteins in the Ras-MAPK and PI3K-Akt signaling cascades.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

15

Thermo Scientific Products for Pre-fractionation and Enrichment

Highlights: • High recovery – equivalent or greater glycoprotein recovery vs. competitor kits and lectin resins • Fast – glycoprotein purification in less than one hour • Versatile – isolate glycoproteins from various sample types; e.g., human serum and cell lysate • Robust – lectin does not leach from resin when processing sample • Convenient – complete kit contains lectin resins and spin columns with all necessary reagents • Compatible with Bradford-based protein assays – dialysis or protein precipitation of recovered glycoproteins is not required before protein assay

A.

Glycoprotein Isolation Kit, ConA

89805

Glycoprotein Isolation Kit, WGA Sufficient reagents to isolate glycoproteins with strong affinity for WGA from 10 samples of up to 640 µl (1-1.5 mg total protein) each Includes: WGA Lectin Resin, 1.1 ml resin supplied as a 50% slurry Binding/Wash Buffer, 5X Stock Solution, 6.5 ml Elution Buffer, 5 ml Column Accessory Pack, 10 Spin Columns with Caps and 20 Collection Tubes

A.

B.

Figure 4. Glycoprotein isolation from human serum and cell lysate: performance comparison of kits using ConA resin. Human serum and CHO lysate samples were processed with the Thermo Scientific Glycoprotein Isolation Kit, ConA and with other commercially available ConA resins. An equivalent amount of total protein was applied to each resin. Eluted glycoprotein fractions were compared with ConA Resin boiled in SDS-PAGE Buffer to release lectins. All fractions were normalized by volume and resolved on 8-16% polyacrylamide gels. Gels were silver-stained. A. Eluted glycoprotein fractions from applied human serum and B. eluted glycoprotein fractions from applied CHO lysate. Arrows identify protein bands that result from ConA leaching from the resin during the elution process.

16

Pkg. Size Kit

Sufficient reagents to isolate glycoproteins with strong affinity for ConA from 10 samples of up to 640 µl (1-1.5 mg total protein) each. Includes: ConA Lectin Resin, 1.1 ml resin supplied as a 50% slurry Binding/Wash Buffer, 5X Stock Solution, 6.5 ml Elution Buffer, 5 ml Column Accessory Pack, 10 Spin Columns with Caps and 20 Collection Tubes

ConA

Supplier G

Supplier A

Thermo Scientific

B.

Product # Description

CHO Lysate

ConA

Supplier G

Supplier A

Thermo Scientific

Figure 5. Glycoprotein isolation from human serum and cell lysate: performance comparison of kits using WGA resin. Human serum and CHO lysate samples were processed with the Thermo Scientific Glycoprotein Isolation Kit, WGA and with other commercially available WGA resins. An equivalent amount of total protein was applied to each resin. Eluted glycoprotein fractions were normalized by volume and resolved on 8-16% polyacrylamide gels. A. Eluted glycoprotein fraction from applied human serum and B. eluted glycoprotein fraction from applied CHO lysate.

Ordering Information

89804 Human Serum

Supplier A

Two lectin-based glycoprotein isolation kits, concanavalin A (ConA) and wheat germ agglutinin (WGA), allow isolation of glycoproteins from complex protein mixtures, including serum, tissue and cultured cell lysates, thus enabling downstream analysis. ConA lectin recognizes α-linked mannose and terminal glucose residues, while WGA lectin selectively binds to N-acetyl glucosamine (GlcNAc) groups and to sialic acid.

CHO Lysate Thermo Scientific

Isolate glycoproteins from complex protein mixtures.

Human Serum Thermo Scientific

Glycoprotein Isolation Kits

For more information, or to download product instructions, visit www.thermo.com/pierce

Kit

The Thermo Scientific Ubiquitin Enrichment Kit isolates polyubiquitin protein conjugates from cultured cells and tissue samples. The enriched fraction is analyzed to determine the amount of general ubiquitin conjugates present or to identify a specific protein by Western blotting. The assay protocol is fast, straightforward and allows isolation of polyubiquitinated proteins and the fractionation of monoubiquitinated species in the resin flow-through. The Ubiquitin Enrichment Kit outperforms other suppliers’ kits and provides a clean and specific preparation of proteins when compared to a control resin. Highlights: • Fast – less than 45 minutes hands-on time • Complete – includes all reagents needed for ubiquitin-modified protein enrichment from cultured cells and tissue samples, including spin columns and ubiquitin antibody • Flexible – sample incubation from 2 hours to overnight allows assay to be completed in several hours or in less than 30 minutes after overnight incubation • Robust – compatible with all Thermo Scientific Cell Lysis Solutions and standard RIPA formulations • Multiple-sample format – easily processes 1-15 samples concurrently

Elution

Flow-through

GSH Resin

Elution

Ab-based Method Flow-through

Elution

Supplier C

Elution

EHela load

The ubiquitin proteasome pathway is the principal non-lysosomal pathway that controls the proteolysis of proteins. This pathway is significantly involved in a variety of cellular processes, including DNA repair, transcriptional regulation, signal transduction, cell metabolism and morphogenesis. Differences in total ubiquitination or the ubiquitination of specific proteins affect numerous pathological conditions, including malignancies, certain genetic diseases and neurodegenerative diseases.1

Flow-through

Thermo Scientific

Recover ubiquitin modified protein in < 45 minutes.

Flow-through

Ubiquitin Enrichment Kit

kDa

220 120 100 80 60 50 40

20

Figure 6. The Thermo Scientific Ubiquitin Enrichment Kit recovers more ubiquitin-modified proteins than any other method. Epoxomicin-treated HeLa cell lysates (EHeLa, 150 µg) were enriched. After elution, all samples were normalized to the initial load (EHeLa load). The flow-through and elution obtained using the Thermo Scientific Ubiquitin Enrichment Kit are shown first. The results using another supplier’s enrichment kit are shown for comparison (Supplier C, manufacturer’s instructions for this kit were followed). Additionally, the results obtained using an anti-ubiquitin monoclonal antibody-based enrichment scheme (antibody-based) and a negative control resin (GSH resin) are shown. The elution from each resin shows the amount of ubiquitin-modified protein that was captured using that method. Reference 1. Ciechanover, A. (1998). The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17(24), 7151-1760.

Ordering Information Product # Description 89899

Ubiquitin Enrichment Kit

Pkg. Size Kit

Contains sufficient materials for enriching up to 15 lysate samples containing ~0.15 mg total protein per sample. Pack 1 Polyubiquitin Positive Control (1,000X), 50 µl, 2 mg/ml Anti-ubiquitin Antibody, 50 µl rabbit antiserum Pack 2 Polyubiquitin Affinity Resin, 300 µl, supplied as a 25% slurry Binding Capacity: ~1 µg per 20 µl of slurry BupH™ Tris Buffered Saline Pack, 1 ea., makes 500 ml of 0.025 M Tris, 0.15 M NaCl; pH 7.2 Spin Columns and Accessories, 18 columns with top and bottom caps

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

17

Thermo Scientific Products for Pre-fractionation and Enrichment Organelle Enrichment Kits

Lamp-1

Enrichment kits for lysosomes, peroxisomes and nuclei. Liver

Subcellular fractionation simplifies complex protein mixtures, thereby facilitating proteomic analysis. We developed three organelle enrichment kits for lysosomes, peroxisomes and nuclei that enable enrichment of intact organelles from cells and tissue. Each kit uses density gradient centrifugation to separate organelles from contaminating cellular structures (Figures 7-11). The isolated organelles are suitable for applications such as 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction and interaction or localization studies. Highlights: • Efficient and easy to use – kit reagents and gradient centrifugation separate organelles from contaminating structures (Table 2) • Compatible – prepare samples for downstream applications, including 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction and interaction or localization studies • Complete – kits contain sufficient material for 25 applications

Kidney

Lysosomes

Lysate

Figure 8. Lysosome enrichment from tissue. Liver and kidney tissues (200 mg each) were processed using the Thermo Scientific Lysosome Enrichment Kit for Tissue and Cultured Cells. Total lysate and isolated lysosomes were analyzed by Western blotting for Lamp-1, a lysosomal membrane protein marker.

PMP70

Liver

Table 2. Thermo Scientific Organelle Enrichment Kits are a convenient and fast means for sample preparation. Target Organelle

Sample Source

Lysosome

Cells

OptiPrep® Density Gradient

Centrifugation Speed (x g)

Centrifugation Time (Mins)

15%, 17%, 20%, 23%, 27% and 30%

145,000

120

Kidney

Tissue (soft & hard) Peroxisome

Soft Tissue

27.5%, 30% and 35%

180,000

90

Hard Tissue

18%, 20% and 27.5%

180,000

90

Nuclei

Tissue (soft & hard)

7%, 23% and 27.5%

40,000

90

Lamp-1

Lysate

Peroxisomes

Figure 9. Peroxisome enrichment from tissue. Liver and kidney tissues (300 mg each) were processed using the Thermo Scientific Peroxisome Enrichment Kit for Tissue. Total lysate and isolated peroxisomes were analyzed by Western blotting for PMP70, a peroxisomal membrane protein marker.

Cathepsin D

Figure 10. Nuclei enrichment from tissue. Liver and kidney tissue (400 mg each) Ab: HDAC A431 cells

Liver

HeLa cells

Lysate

Lysosomes

Lysate

Lysosomes

Figure 7. Lysosome enrichment from cultured cells. A431 and HeLa wet cell paste (~200 mg) was processed using the Thermo Scientific Lysosome Enrichment Kit for Tissue and Cultured Cells. Total cell lysate and isolated lysosomes were analyzed by Western blotting for Lamp-1 and Cathepsin D, membrane-bound and soluble lysosome markers, respectively.

Kidney

Lysate

Nuclei

were processed using the Thermo Scientific Nuclei Enrichment Kit for Tissue. Total cell lysate and isolated nuclei were analyzed by Western blotting for histone deacetylase (HDAC), a soluble nuclear marker.

18

For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific COX4 PMP70

Supplier S COX4 PMP70

Liver

Ordering Information Product # Description

Lysosomes

89839

Kidney

Lysosome Enrichment Kit for Tissues and Cultured Cells Sufficient material for 25 applications. Includes: Lysosome Enrichment Reagent A Lysosome Enrichment Reagent B OptiPrep Cell Separation Media BupH Phosphate Buffered Saline

Liver Nuclei

89840

Kidney

Peroxisome Enrichment Kit for Tissue Sufficient material for 25 applications. Includes: Peroxisome Enrichment Reagent A Peroxisome Enrichment Reagent B OptiPrep Cell Separation Media BupH Phosphate Buffered Saline

Liver Peroxisomes

89841 Kidney

Figure 11. Cross-contamination of enriched organelles. Liver and kidney tissues (200 mg each) were processed using each organelle isolation kit and analyzed via Western blotting for contamination by probing for COX4 and PMP70, mitochondria and peroxisome markers, respectively. Samples were normalized by protein amount (~ 10 µg of total proteins), except for the liver and kidney fractions from the peroxisome experiment, which were normalized by volume. Tissues from rats of identical weight and ages were processed with similar kits from Supplier S according to the manufacturer’s instructions. Significant contamination in the lysosome and nuclei fractions is observed with the procedure from Supplier S. References Gjoen, T., et al. (1997). Lysosomes and endocytosis. Subcellular fractionation: A practical approach. Eds. Graham, J.M. and Rickwood, D., IRL Press Limited, Oxford, England, 169-200. Graham, J.M. (1997). Homogenization of tissues and cells. Subcellular fractionation: A practical approach. Eds. Graham, J.M. and Rickwood, D., IRL Press Limited, Oxford, England, 1-28. Hajra, A.K., et al. (1985). Anal Biochem, 148(2), 233-244. Hinton, R.H. and Mullock, B.M. (1997). Isolation of subcellular fractions. Subcellular fractionation: A practical approach. Eds. Graham, J.M. and Rickwood, D., IRL Press Limited, Oxford, England, 31-69. Luers, G., et al. (1993). J. Cell. Bio., 121(6), 1271-1280. Lukong, K.E., et al. (2001). J. Biol Chem., 276, 46172-46181. Van Veldhoven, P.P., et al. (1996). Anal Biochem, 237(1), 17-23.

Nuclei Enrichment Kit for Tissue Sufficient material for 25 applications. Includes: Nuclei Enrichment Reagent A Nuclei Enrichment Reagent B OptiPrep Cell Separation Media BupH Phosphate Buffered Saline

89874

Mitochondria Isolation Kit for Cultured Cells† Sufficient reagents for 50 applications. Includes: Mitochondria Isolation Reagent A Mitochondria Isolation Reagent B Mitochondria Isolation Reagent C

89801

Mitochondria Isolation Kit for Tissue† Sufficient reagents for 50 applications. Includes: Mitochondria Isolation Reagent A Mitochondria Isolation Reagent B Mitochondria Isolation Reagent C Bovine Serum Albumin BupH Phosphate Buffered Saline

Pkg. Size Kit 90 ml 90 ml 50 ml 1 pack

Kit 90 ml 90 ml 50 ml 1 pack

Kit 90 ml 90 ml 50 ml 1 pack

Kit 50 ml 500 ml 70 ml

Kit 50 ml 500 ml 65 ml 230 mg 1 pack

Complementary Products 89910

HDAC Polyclonal Antibody

50 µg

89911

PMP 70 Polyclonal Antibody

50 µg

89912

Beta’-COP Polyclonal Antibody

50 µg

89913

VDAC Polyclonal Antibody

50 µg

89915

Cathepsin Monoclonal Antibody

25 µg

89916

Nucleoporin p62 Monoclonal Antibody

25 µg

89917

Lamp-1 Monoclonal Antibody

50 µg

89918

Cytochrome C Monoclonal Antibody

100 µg

† See patent information.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

19

Thermo Scientific Products for Pre-fractionation and Enrichment Mammalian cells (2 x 107)

Mitochondria Isolation Kit for Cultured Cells

Add 800 µl Reagent A Option A. Add 10 µl Incubate Reagent B 2 minutes on ice

Isolate intact mitochondria with maximum yield in only 40 minutes.

Option B. Dounce homogenize

Highlights: • Fast – isolate intact mitochondria in approximately 40 minutes • Multi-sample format – reagent-based method allows for concurrent preparation of multiple samples • Optional alternate method – reagents and protocol included for traditional Dounce homogenization • Benchtop-compatibility – isolation performed in a microcentrifuge tube The isolation of mitochondria is typically a laborious process requiring single-sample processing with Dounce homogenization. The Thermo Scientific Mitochondria Isolation Kit for Cultured Cells uses a non-mechanical, reagent-based method (Figure 12, Option A) that allows multiple samples (≤ 6) to be processed concurrently. Cultured mammalian cell pellets are gently lysed using a proprietary formulation that results in maximum yield of mitochondria with minimal damage to integrity. The kit also offers a second isolation method based on traditional Dounce homogenization (Figure 12, Option B), which results in two-fold more mitochondria recovery, as determined by protein assay. Both methods use differential centrifugation to separate the mitochondrial and cytosolic fractions with a bench-top microcentrifuge and are completed in approximately 40 minutes (post-cell harvest). Once isolated, the mitochondria can be used in downstream applications such as apoptosis, signal transduction and metabolic studies, as well as to facilitate mitochondrial proteomics efforts.

Incubate 5 minutes on ice Vortex every minute

Add 800 µl Reagent C Centrifuge 700 x g 10 minutes at 4°C

Pellet (Nuclei and cell debris)

Supernatant *Centrifuge 12,000 x g 15 minutes at 4°C

Cytosol

Mitochondria Wash with 500 µl Reagent C

Centrifuge 12,000 x g 5 minutes at 4°C

Figure 12. Procedure for the isolation of mitochondria from cultured mammalian cells using Option A. the reagent-based method and Option B. the Dounce-based method. * A more purified preparation of mitochondria can be obtained by centrifuging at 3,000 x g instead of 12,000 x g.

The reagent- and Dounce-based isolation procedures are outlined in Figure 12. Approximately 2 x 107 mammalian cells (NIH 3T3 or C6) were pelleted per sample in a 2 ml microcentrifuge tube. The cells were resuspended in Mitochondria Isolation Reagent A and incubated on ice for 2 minutes. Using the reagent-based method, the cells were lysed by adding Mitochondria Isolation Reagent B in conjunction with frequent vortexing. The lysate was mixed with Mitochondria Isolation Reagent C and centrifuged to remove nuclei, unbroken cells and cellular debris. The supernatant was subsequently centrifuged to collect the mitochondria, and the pellet was surface-washed to remove cytosolic contaminants. Mitochondria prepared using Dounce homogenization followed a similar protocol. Briefly, 2 x 107 NIH 3T3 cells were resuspended in Mitochondria Isolation Reagent A, incubated for 2 minutes and then transferred to a tissue grinder and lysed with 80 strokes. The cell homogenate was mixed with Mitochondria Isolation Reagent C and the remainder of the protocol as detailed in Figure 12, Option B was performed. Damage to the outer membrane was assessed by Western blot analysis of Cytochrome C and voltage-dependent anion channel (VDAC) (Figure 13). Cytochrome C resides in the intermembrane space of undamaged mitochondria and VDAC is an integral membrane protein in the outer mitochondrial membrane. Negligible amounts of both proteins were present in the cytosol, indicating that the mitochondria remained intact during isolation. Contamination of the mitochondria with cytosolic components was negligible. Following a single wash of the collected pellet of mitochondria, minimal heat shock protein 90 (Hsp90) contamination was detected in a Western blot (Figure 14).

20

For more information, or to download product instructions, visit www.thermo.com/pierce

Cytochrome C M

Table 3. Collection of mitochondria (reagent-based method).

VDAC C

M

C

Reagentbased A.

B.

C.

D.

RCF (x g)

Protein (µg)

% Total Protein

3,000

159.1

62

12,000

98.2

38

Once isolated, mitochondria are ready for many downstream applications, including Western blotting. 2-D Western analysis of isolated mitochondria was used to identify Mn-superoxide dismutase, a detoxifying enzyme residing in the mitochondrial matrix (Figure 16).

Douncebased

Figure 13. Analysis of mitochondrial integrity. Mitochondria and cytosol fractions were prepared from C6 cells using the reagent-based method (A. and B.) or Dounce homogenization (C. and D.) Fractions were analyzed via Western blot for cytochrome C (A. and C.) or voltage-dependent anion channel (VDAC) (B. and D.). Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Product # 34080) was used for detection. M = mitochondria and C = cytosol.

3

pl

MW (K) 222 — 114 — 88 — 50 —

10

33 — 27 —

Protein

Location

hsp90

Cytosol

M

C 17 —

Figure 14. Analysis of cytosolic contamination in mitochondria fraction. Mitochondria and cytosol fractions were prepared from NIH 3T3 cells. Each fraction was analyzed by Western blot for the cytosolic protein, Hsp90. M = mitochondria and C = cytosol.

A more purified preparation of mitochondria was obtained by decreasing the centrifugation speed used to collect the organelle. Mitochondria collection, normally performed at 12,000 x g, was split into a low-speed collection at 3,000 x g and a subsequent high-speed collection of remaining mitochondria in the supernatant at 12,000 x g. Western blot analysis of PMP70, an abundant integral membrane protein of the peroxisome, and Cathepsin S, a cysteine protease in the lysosome, resulted in > 50% reduction in contamination from these organelles (Figure 15) while recovering approximately 60% of the mitochondria routinely collected with a higher centrifugation speed (Table 3).

A.

Protein

Location

PMP70

Peroxisome

B. Cathepsin S

M1

M2

C

M1

M2

C

Figure 16. 2-D Western blot of superoxide dismutase (Mn-SOD) in isolated mitochondria. Mitochondria were isolated from NIH 3T3 cells using the Dounce method, resolved by 2-DE and analyzed by Western blot for manganese-containing SOD. Approximately 15 µg of mitochondrial protein was focused on an 11 cm, pH 3-10 IPG strip. The second dimension was performed using 8-16% SDS-PAGE.

Ordering Information Product # Description 89874

89918

Pkg. Size

Mitochondria Isolation Kit for Cultured Cells

Kit

Sufficient reagents for 50 applications. Includes: Mitochondria Isolation Kit Reagent A Mitochondria Isolation Kit Reagent B Mitochondria Isolation Kit Reagent C

50 ml 500 µl 70 ml

Cytochrome C Monoclonal Antibody

100 µg

Lysosome

Figure 15. Reduction of lysosomal and peroxisomal contaminants in mitochondrial fraction. Mitochondria and cytosol fractions were prepared using a modified reagent-based isolation method. Heavy, more purified mitochondria were collected at 3,000 x g and the supernatant was centrifuged at 12,000 x g to collect remaining mitochondria. Each fraction was analyzed by Western blot for A. Peroxisomal membrane protein 70 (PMP70, C6 cells) and B. Lysosomal Cathepsin S (NIH 3T3 cells). M1 = 3,000 x g mitochondria fraction, M2 = 12,000 x g mitochondria fraction and C = cytosol **See Table 3 for protein quantification.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

21

Thermo Scientific Products for Pre-fractionation and Enrichment Mitochondria Isolation Kit for Tissue

MW (K)

Isolate intact mitochondria with maximum yield in only 60 minutes. Highlights: • Quick and convenient – isolates intact mitochondria in less than 60 minutes • Versatile – isolate soft- and hard-tissue samples • Multi-sample format – reagent-based approach enables simultaneous processing of multiple samples • Optional alternate method – reagents and protocol included for the traditional Dounce homogenization procedure with fewer required strokes • Bench-top-compatible – both procedures are performed using a microcentrifuge tube The Thermo Scientific Mitochondria Isolation Kit for Tissue (Product # 89801) isolates intact mitochondria from soft- and hard-tissue samples. The kit offers two methods for mitochondria isolation. The first method uses a unique reagent-based procedure that enables simultaneous multi-sample processing. The second method relies on traditional Dounce homogenization for tissue disruption and subsequent isolation. Both procedures use differential centrifugation to separate the intact mitochondria using a benchtop microcentrifuge and are completed in less than 60 minutes. In addition, both procedures have been optimized for maximum yield of mitochondria with minimal damage to its integrity (Figure 17). The isolated mitochondria can be used for many applications, including 1-D and 2-D Western blotting (Figure 18) and protein profiling by mass spectroscopy. COX4 M

Reagentbased 1.

VDAC C

M 2.

M

5.

32.1 — 26.1 — 16.1 —

Figure 18. 2-D Western blot of superoxide dismutase (Mn-SOD) in isolated mitochondria. Intact mitochondria from the liver of a female Sprague-Dawley rat was processed using the Dounce homogenization method. The isolated mitochondria was lysed using Thermo Scientific M-PER Mammalian Protein Extraction Reagent (Product # 78501) and approximately 35 µg of total mitochondrial protein was added to 2-D sample buffer (8 M urea, 4% CHAPS, pH 5-8 carrier ampholytes, 50 mM DTT). Proteins were resolved on a pH 5-8 IPG strip followed by 8-16% SDS-PAGE and analyzed by Western blot for Mn-SOD. References for Mitochondrial Proteome 1. Lescuyer, P., et al. (2003). Progress in the definition of a reference human mitochondrial proteome. Proteomics 3, 157-167. 2. Taylor, S.W., et al. (2003). Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 21, 281-285.

Ordering Information Product # Description 89801

C

3.

Pkg. Size

Mitochondria Isolation Kit for Tissue

Kit

Sufficient reagents for 50 isolations of intact mitochondria from soft and hard tissue. Includes: Mitochondria Isolation Kit Reagent A Mitochondria Isolation Kit Reagent B Mitochondria Isolation Kit Reagent C BSA BupH Phosphate Buffered Saline

50 ml 500 µl 65 ml 235 mg 1 pack

Cytochrome C Monoclonal Antibody

6.

Figure 17. Analysis of mitochondrial integrity. Mitochondria (M) and cytosolic (C) fractions were prepared from fresh rat liver (Panels 1, 2, 3, 5 and 6) and heart (Panel 4) tissue samples using the reagent-based and Dounce homogenization methods. Fractions were analyzed via Western blot for COX4, voltage-dependent anion channel (VDAC) and Cytochrome C. Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Product # 34080) was used for detection. COX4 is an inner-mitochondria membrane protein, VDAC is an outer-mitochondria membrane protein and cytochrome C is located in the intermembrane space.

22

10

49.3 —

89918 Douncebased 4.

pl

220 — 114 — 80.2 —

Cytochrome C C

3

For more information, or to download product instructions, visit www.thermo.com/pierce

100 µg

Thermo Scientific Products for Sample Clean-up

High-Performance Dialysis Product Selection Guide 10-100 µl

0.1-30 ml

15-100 ml

MWCO Membrane

Thermo Scientific Slide-A-Lyzer® MINI Dialysis Unit†

Thermo Scientific Slide-A-Lyzer Dialysis Cassette†

Thermo Scientific SnakeSkin® Dialysis Tubing

2K

N/A

•

N/A

3.5K

•

•

•

7K

•

•

•

10K

•

•

•

20K

•

•

N/A

† See patent information.

High-Performance Dialysis

Slide-A-Lyzer MINI Dialysis Units

Our dialysis products take the hassle and the mess out of sample dialysis.

For sample volumes as small as 10 µl.

The Thermo Scientific High-performance Dialysis Products are ideally suited for the variety of sample sizes common in research laboratories. The different MINI Units, Cassettes and Tubing products pictured here are available in 2K, 3.5K, 7K, 10K and 20K molecular-weight cutoff membrane options (MWCO, denoted by blue, pink, green, orange and purple labels and cassette colors, respectively). 12-30 ml

0.5-3 ml

Highlights: • 100% leak-tested – patented design does not permit “wicking” that can occur in homemade devices • Very affordable • Excellent sample recoveries – the Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit generally recovers 9-10 µl after dialysis of a 10 µl sample • Time of dialysis drastically reduced – converts 100 µl of pH 2.8 buffer to pH 9.4 dialyzing against 1 L bicarbonate buffer, pH 9.4 in less than 10 minutes

3-12 ml 0.1-0.5 ml

10-100 µl Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit

1. Apply sample with a pipette.

2. Place the Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit into the float.

3. Insert the float into the beaker containing the dialysate.

4. Recover sample.

15-100 ml Thermo Scientific SnakeSkin Dialysis Tubing

Figure 1. Four-step procedure for buffer exchange using Thermo Scientific Slide-A-Lyzer MINI Dialysis Units.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

23

Thermo Scientific Products for Sample Clean-up Slide-A-Lyzer Dialysis Cassettes Require just half the time of dialysis tubing! The Thermo Scientific Slide-A-Lyzer Dialysis Cassette is the product of choice for rapidly dialyzing sample volumes from 100 µl to 30 ml. Highlights: • > 95% sample recovery – sample volume remains visible throughout dialysis • No knots or clamps to loosen and leak – secure design prevents sample loss due to leaks • Rigid frame permits smooth sample withdrawal of submilliliter volumes – removing every last drop is easy – even for scientists who have never before performed dialysis • High surface area/sample volume ratio will dialyze twice as fast as dialysis via conventional tubing – patented Cassette design spreads the sample over a large surface area and the double membrane promotes fast dialysis • Clear color-coded plastic allows you to see your injection

1. Remove a Cassette from the protective pouch. Fill the Cassette cavity with your sample through one of the guide inlets in the corner of the cassette. With the syringe still inserted into the cavity, draw up on the syringe to remove air.

2. Attach a flotation buoy and dialyze. Each buoy serves as an effective flotation device and also as a convenient bench-top stand for the Cassette. There is no need to worry about suspension of the dialysis bag.

Updated dialysis and desalt brochure! Request a copy of the updated Dialysis and Desalt Handbook that features the following products: • Slide-A-Lyzer MINI Dialysis Units with 3.5K, 10K, and 20K membranes for highrecovery dialysis of 100-100 µl samples • Slide-A-Lyzer Dialysis Cassettes with 2K, 3.5K, 7K, 10K and 20K membranes for high-recovery dialysis of 0.1-30 ml samples • SnakeSkin Dialysis Tubing, made with regenerated cellulose with 3.5K, 7K and 10K MWCO for quick, clean and easy dialysis of 15-100 ml samples • Zeba Protein Desalt Products

3. Inject the Cassette chamber with air and withdraw your dialyzed sample from the Cassette. Figure 2. Use of a Thermo Scientific Slide-A-Lyzer Dialysis Cassette.

Visit our website www.thermo.com/dialysis for information and ordering information for the complete Slide-A-Lyzer Product Line.

Log on to our website or contact your distributor to request a copy.

24

For more information, or to download product instructions, visit www.thermo.com/pierce

Zeba Desalt Spin Columns Desalt sample volumes ranging from 2 µl to 4 ml and experience exceptional protein recovery quickly.

Although numerous techniques and resins for desalting are available, most have many drawbacks, including significant sample loss, long processing times and the need to collect multiple fractions. Thermo Scientific Zeba Desalt Spin Columns provide excellent protein recovery without the limitations associated with other desalting methods. Zeba Desalt Spin Columns are available in micro†, 0.5, 2, 5 and 10 ml formats and allow processing of samples ranging from 2 µl to 4 ml (Table 1). Table 1. Recommended sample volumes for Thermo Scientific Zeba Spin Columns. Resin Bed

Sample Volume

75 µl (micro) column

2-12 µl

0.5 ml column

30-130 µl

2 ml column

200-700 µl

5 ml column

600-2,000 µl

10 ml column

1,500-4,000 µl

96-well

20-100 µl

Zeba Desalt Spin Columns contain a proprietary and exclusive high-performance desalting resin that offers exceptional desalting and protein-recovery characteristics compared to other commercially available resins (Figure 3). Samples containing as low as 25 µg/ml of protein can be processed, providing exceptional protein recovery and ≥ 95% retention of salts and other small molecules (< 1,000 MW).

Supplier G

Supplier B

Control

Thermo Scientific

Sample Size 200 µl + 50 µl stacker

Supplier G

Supplier B

Thermo Scientific

Sample Size 700 µl

Control

Highlights: • Exceptional protein recovery • Wide product offering accommodates your sample needs • Easy to use with no cumbersome column preparation or equilibration • No screening fractions for protein or waiting for protein to emerge by gravity flow • Minimal sample dilution

Sample 250 µg/ml BSA (66 kDa)

A.

5.2

97%

78%

79%

0.19

0.45

0.73

5.2

90%

56%

29%

Recovery

0.14

0.07

0.14

Conductivity (mho) Sample 25 µg/ml BSA (66 kDa)

B.

Figure 3. Increased protein recovery with Thermo Scientific Zeba Desalt Spin Columns. Samples of bovine serum albumin (BSA) at A. 250 µg/ml and B. 25 µg/ml in 1 M NaCl were desalted with the 2 ml Zeba Desalt Spin Columns and other commercial desalting resins using similar formats. A portion of the recovered sample (10 µl) was analyzed by SDS-PAGE. The remaining sample was used for conductivity measurements and the Thermo Scientific Pierce BCA Protein Assay (Product # 23225) was performed to determine protein concentration. Zeba Desalt Resin provides significantly greater protein recovery under all conditions tested. Conductivity and protein recovery values after desalting are indicated for 250 µg/ml samples.

Ordering Information Product # Description

Pkg. Size

89877

Zeba Micro Desalt Spin Columns†

25/pkg.

89878

Zeba Micro Desalt Spin Columns

50/pkg.

89882

Zeba Desalt Spin Columns, 0.5 ml

25/pkg.

89883

Zeba Desalt Spin Columns, 0.5 ml

50/pkg.

89889

Zeba Desalt Spin Columns, 2 ml

5/pkg.

89890

Zeba Desalt Spin Columns, 2 ml

25/pkg.

89891

Zeba Desalt Spin Columns, 5 ml

5/pkg.

89892

Zeba Desalt Spin Columns, 5 ml

25/pkg.

89893

Zeba Desalt Spin Columns, 10 ml

5/pkg.

89894

Zeba Desalt Spin Columns, 10 ml

25/pkg.

89807

Zeba 96-well Desalting Spin Plates

2 Plates.

89808

Zeba 96-well Desalting Spin Plates

4 Plates.

Spin Columns (No Resin) 89879

Pierce Centrifuge Micro Spin Columns

50/pkg.

89868

Pierce Centrifuge Columns, 0.8 ml

50/pkg.

89896

Pierce Centrifuge Columns, 2 ml

25/pkg.

89897

Pierce Centrifuge Columns, 5 ml

25/pkg.

89898

Pierce Centrifuge Columns, 10 ml

25/pkg.

† See patent information.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

25

Thermo Scientific Products for Sample Clean-up

Eliminate band distortions caused by contaminants. Highlights: • Eliminates artifacts caused by incompatible contaminants – removes dyes, reducing agents, detergents, sugars, glycerol, guanidine, urea and ammonium sulfate to provide reproducible results on SDS-PAGE analysis • Compatible with the Thermo Scientific Pierce BCA Protein Assay – allows quantification of the processed sample • Enriches dilute protein solutions – concentrates protein sample by eight-fold in less than 20 minutes • Fast and easy-to-use for up to 70 µg of protein per sample – uses new spin-cup format that allows processing of high amounts of protein The Thermo Scientific Pierce SDS-PAGE Sample Prep Kit improves SDS-PAGE analysis of difficult-to-analyze samples. While numerous compounds are incompatible with typical electrophoretic running buffers and SDS-PAGE separation, our kit cleans up protein samples in minutes, removing a wide range of interfering chemicals, such as chaotropic agents, detergents, lipids, pH extremes and salts. Untreated M

S

Thermo Scientific - Treated M

S

M

S

M

Prepare the following sample types for SDS-PAGE analysis: • Inclusion bodies solubilized in guanidine•HCl • Samples containing low-pH buffers, thiocyanate or urea • Proteins precipitated in ammonium sulfate • Dilute protein solutions 100 Percent Protein Recovered

Pierce SDS-PAGE Sample Prep Kit

88%

80

85% 77%

75%

77%

40 20 0 Carbonic Anhydrase

Ovalbumin

Transferrin

Ubiquitin Cytochrome C Bacterial Lysate

Figure 5. Consistent protein recovery is achieved. Pure proteins (60 µg) of assorted molecular weights from left to right: 30K, 44K, 80K, 86K and 12K and bacterial lysate at 27K were processed using this kit. Protein concentrations were determined with the Thermo Scientific Pierce BCA Protein Assay and reported as percent protein recovered. Table 2. Compatibility of common SDS-PAGE-interfering reagents.

S

Interfering Reagents

Figure 4. Eliminate distortion caused by detergents. Rat C6 cells were lysed and a membrane protein fraction isolated using Thermo Scientific Mem-PER Eukaryotic Membrane Protein Extraction Reagent (Product # 89826). Membrane and hydrophilic cell fractions were separated by SDS-PAGE using 4-20% gradient gels with or without prior treatment using our kit. Western blot analysis was performed using an antibody against cytochrome oxidase subunit 4 (COX 4) and Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate (Product # 34095). The kit-treated samples exhibit better band straightness and resolution with low molecular weight proteins than samples that were untreated. S = Soluble fraction (hydrophilic) and M = Membrane fraction.

Percent Protein Recovered (Starting amount = 20 µg BSA)

Control (Water)

75%

0.5 M Sodium Chloride

80%

2 M Ammonium Sulfate

76%

20% SDS

75%

10% Triton® Detergent

75%

6 M Urea: DMSO (1:3 ratio)

75%

1M Sodium Chloride

75%

6M Urea

74%

10% CHAPS

80%

25% Glycerol

71%

10% OTG

71%

2 M Guanidinium•HCl

70%

40% Sucrose

70%

Ordering Information Product # Description 89888

26

74%

60

Pkg. Size

Pierce SDS-PAGE Sample Prep Kit

Kit

Sufficient reagents for 50 2-300 µl samples. This product replaces Product # 26800. Includes: Protein Binding Resin Elution Buffer Purified DMSO Spin Cups Collection Tubes

1 ml 5.0 ml 27 ml 50 72

For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Products for Protein Detection and Gel Separation SuperSignal Chemiluminescent Substrates† A sensitive, user-friendly detection system for 2-D Western blotting.

MW (K) 116 — 84 —

5

pl

8

pl

10

48 —

Methods Membrane protein extracts were prepared from cultured NIH 3T3 or S. cerevisiae EGY194 cells using the 2-D-Sample Prep for Membrane Proteins Kit (Product # 89864). Nuclear protein extracts were prepared from TNFα-induced HeLa cells using the 2-D Sample Prep for Nuclear Proteins Kit (Product # 89863). Approximately 15-30 µg of purified protein was processed and focused on 11 cm pH 5-8 or pH 3-10 IPG strips. The second dimension was performed using 8-16% SDS-PAGE, and the resulting 2-D gels were transferred by electroblotting to nitrocellulose membranes for Western blot analysis. The nonspecific sites were blocked overnight using Thermo Scientific SuperBlock (TBS) Blocking Buffer (Product # 37535) containing 0.05% Tween-20 at 4°C with shaking. The blots were incubated with primary antibodies against the integral membrane proteins flotillin and mitochondrial porin, and the nuclear proteins NF-κB:p50 and p65 for one hour at room temperature (RT). The blots were then washed three times with TBS (Product # 28376) containing 0.05% Tween -20. Following the wash, the blots were incubated in HRP-conjugated secondary antibodies (Product #s 31402, 31430 and 31460) for one hour at RT. After washing with TBS (Product # 28376) containing 0.1% Tween -20, the blots were incubated for five minutes in SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Product # 34095). Excess substrate was drained and the blots were exposed to Thermo Scientific CL-XPosure™ Film (Product # 34090) for 1 second. Results and discussion Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate was highly sensitive and effective for detecting proteins in both low and high abundance in complex protein mixtures resolved on 2-D gels. The integral membrane proteins flotillin and mitochondrial porin were identified in mammalian and yeast membrane protein extracts, respectively (Figure 1). Similar sensitivity also occurred with transcription factors stimulated to translocate from the cytosol to the nucleus in response to TNFα. NF-κB p50 and its precursor, p105, were readily detected in nuclear extracts prepared from TNFα-stimulated cells (Figure 2A). Degradation of the p50 factor was also seen that is likely from inhibitor IkB loss. Another NF-κB transcription factor, p65, was also identified (Figure 2B). Conclusion SuperSignal West Femto Chemiluminescent Substrate’s enhanced sensitivity and signal duration allows for an improved signal-tonoise ratio that requires less sample, faster signal generation and greater light emission longevity.

31 — 25 —

A. Flotillin 3

MW (K) 116 — 84 — 48 — 31 — 25 —

B. Mitochondrial Porin

Figure 1. 2-D Western blots of membrane proteins prepared with the Thermo Scientific 2-D Sample Prep for Membrane Proteins Kit. Membrane protein extract was prepared from mammalian or yeast cells and then concentrated and cleaned up. Processed membrane protein samples (approximately 30 µg each as determined by parallel Thermo Scientific Pierce BCA Assay of 2-D SDS-PAGE Sample Prep Resin eluate) were resolved on 2-D gels and analyzed by Western blot for A. flotillin (48 kDa, pI 6.46-6.96, NIH 3T3 cells) and B. mitochondrial porin (30 kDa, pI 6.58-7.08, S. cerevisiae EGY194 cells).

MW (K) 116 — 84 —

3

pl

10

48 — 31 —

A. NF-κB p105 & p50

MW (K) 84 — 48 —

3

pl

10

31 — 25 —

B. NF-κB p65

Figure 2. 2-D Western blots of nuclear proteins prepared with the Thermo Scientific 2-D Sample Prep for Nuclear Proteins Kit. Nuclear protein extract was prepared from TNFα-induced HeLa cells and then desalted/bufferexchanged. Two-dimensional gels were prepared with 15 µg of protein each and analyzed by Western blot for A. NF-κB p105 and p50 and B. NF-κB p65.

Ordering Information Product # Description

Pkg. Size

34080

SuperSignal West Pico Chemiluminescent Substrate

500 ml

34075

SuperSignal West Dura Extended Duration Substrate

100 ml

34095

SuperSignal West Femto Maximum Sensitivity Substrate

100 ml

89863

2-D Sample Prep for Nuclear Proteins

Kit

89864

2-D Sample Prep for Membrane Proteins

Kit

† See patent information.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

27

Thermo Scientific Products for Protein Detection and Gel Separation Novel 2-D Sample Preparation Kits and Other Tools The mechanics of preparing gels have improved greatly over the years; however, sample preparation remains a bottleneck. Problems typically include charged contaminants, dilute solutions, insoluble proteins and low-abundant proteins. Furthermore, common methods to remove contaminants and concentrate proteins, such as dialysis and precipitation, are time-consuming and can be unreliable. We have developed four 2-D sample preparation kits to address these challenges. The kits use protein desalting spin columns to remove small charged contaminants and concurrently buffer-exchange proteins into 2-D Sample Buffer, thereby increasing the amount of protein that can be loaded onto an immobilized pH gradient (IPG) strip. The Thermo Scientific 2-D Sample Prep for Membrane Proteins and the 2-D Sample Prep for Nuclear Proteins combine the isolation of membrane and nuclear proteins, respectively, with sample preparation. Alternatively, the 2-D Sample Preps for Soluble and Insoluble Proteins are for researchers who require clean up of samples isolated by their own methods.

MW (K)

5

pl

8

pl

8

97 — 66 — 43 —

29 — 20 — 14 —

A. Processed

MW (K)

5

97 — 66 — 43 —

29 — 20 — 14 —

2-D Sample Prep for Membrane Proteins Kit B. Unprocessed

Cell fractionation and sample preparation are key for optimal 2-D gel analysis, but are challenging when working with membrane proteins. The Thermo Scientific 2-D Sample Prep for Membrane Proteins Kit combines membrane protein extraction with 2-D sample preparation in a fast, convenient and reliable protocol. Membrane proteins are first extracted from cultured cells using the Thermo Scientific Mem-PER Eukaryotic Membrane Protein Extraction Kit which works on hard or soft tissue samples.3 The prepared extract is subsequently cleaned up in a two-step process. Isolated membrane proteins are first treated with Thermo Scientific Pierce 2-D SDS-PAGE Resin, a modified form of diatomaceous earth that selectively binds to proteins. The resulting eluate is desalted/buffer-exchanged and electrophoresed. Multiple chaotropes present in the 2-D Sample Buffer improve protein solubility1,2 and result in high-resolution 2-D gels. The kit effectively isolates integral membrane proteins from both cultured mammalian and yeast cells (Figure 3) and concentrates proteins up to six-fold, improving detection of low-abundant proteins (Figure 3). Approximately 30-100 µg of membrane proteins are ready for analysis in less than 90 minutes. Highlights: • Requires no precipitation – eliminates difficult resolubilization steps • Isolates efficiently – isolates 30-100 µg of membrane proteins • Concentrates and cleans up – membrane proteins are concentrated up to six-fold and excess detergent is removed • Buffer-exchanges membrane proteins into 2-D Sample Buffer – maintaining proteins in solution • Works quickly – membrane proteins are prepared in less than one hour and are cleaned up in 30-40 minutes • Contains thiourea in the 2-D Sample Buffer – increases protein solubility and improves protein resolution

28

Figure 3. Concentration of membrane protein extract prepared with the Thermo Scientific 2-D Sample Prep for Membrane Proteins Kit. Membrane protein extract was prepared from C6 cells. A. Approximately 75 µl of extract (representing approximately four times the volume shown in B) was processed and buffer-exchanged. The entire sample obtained (35 µg as determined by parallel Thermo Scientific Pierce BCA Assay of 2-D SDS-PAGE Resin eluate) was analyzed on a 2-D gel. B. Approximately 18.5 µl of unprocessed membrane protein extract, corresponding to the maximum volume recommended for IPG strip rehydration, was analyzed directly on a 2-D gel. Both samples were focused on 11 cm, pH 5-8 IPG strips followed by 8-16% SDS-PAGE and then silver stained. References 1. Rabilloud, T., et al. (1997). Electrophoresis 18, 307-316. 2. Lanne, B., et al. (2001). Proteomics 1, 819-828. 3. Benton, B., et al. (2004). A better alternative to Dounce homongenization for the isolation of mitochondria. Previews 8(1), 1-3.

Ordering Information Product # Description 89864

2-D Sample Prep for Membrane Proteins Kit Sufficient material for 25 applications. Includes: Mem-PER Eukaryotic Membrane Protein Extraction Kit: Mem-PER Cell Lysis Reagent Mem-PER Buffer Mem-PER Membrane Protein Solubilization Reagent 2-D Sample Buffer for Membrane Proteins: 2-D Sample Buffer for Membrane Proteins Component A 2-D Sample Buffer for Membrane Proteins Component B 2-D Pierce SDS-PAGE Protein Clean-up and Enrichment Kit: 2-D Pierce SDS-PAGE Protein Binding Resin Binding/Wash Buffer Stock Solution Protein Desalting Spin Columns Elution Buffer Spin Cups Collection Tubes

For more information, or to download product instructions, visit www.thermo.com/pierce

Pkg. Size Kit

5 ml 12.5 ml 20 ml

18 ml 16.5 g

0.5 ml 50 ml DMSO 25 columns 1.25 ml 25 cups 50 tubes

2-D Sample Preparation for Nuclear Proteins Preparation of nuclear proteins for 2-D gel electrophoresis (2-DE) can be time-consuming and frustrating as nuclear proteins are prone to aggregation both in solution and during isoelectric focusing.1 We developed the Thermo Scientific 2-D Sample Prep for Nuclear Proteins Kit to circumvent these issues. This kit combines nuclear protein extraction, using the popular Thermo Scientific NE-PER Nuclear and Cytoplasmic Reagents, with a fast and reliable protocol for 2-D sample preparation. Once isolated, nuclear proteins are buffer exchanged directly into a specially formulated 2-D Sample Buffer using equilibrated protein desalting columns. This procedure eliminates salts that interfere with isoelectric focusing (Figure 4A), while multiple chaotropes1,2 maintain the solubility of nuclear proteins, resulting in minimal sample loss and high-resolution 2-D gels (Figure 4B). Nuclear proteins from a variety of mammalian cell lines and tissues are recovered with ≥ 90% efficiency, while ≥ 98% of salt contaminants are removed. Moreover, as processed samples are recovered in 2-D Sample Buffer, they can be immediately applied to an IPG strip in their entirety, effectively increasing the allowable sample load volume. The 2-D Sample Prep for Nuclear Proteins Kit is a significant improvement over standard precipitation methods that require lengthy incubation and resolubilization steps, often introducing contaminants that can partially obscure 2-D gels (Figure 4C). Highlights: • Removes small charged contaminants that interfere with 2-D electrophoresis – reduces the time for isoelectric focusing and prevents loss of data on 2-D gels due to salt fronts • Buffer-exchanges proteins into 2-D Sample Buffer, increasing amount of protein that can be applied to an IPG strip while maintaining solubility throughout the desalting process • Uses NE-PER Nuclear and Cytoplasmic Reagents – prepares a highly purified nuclear protein extract • Protein extraction and 2-D sample preparation are combined for fast and efficient protocol • Thiourea in sample buffer increases protein solubility and improves protein resolution on 2-D gels1,2 • Multiple samples can be processed in less than 15 minutes References 1. Rabilloud, T., et al. (1997). Electrophoresis 18, 307-316. 2. Lanne, B., et al. (2001). Proteomics 1, 819-828.

4

MW (K)

pl

7

97 — 66 — 43 —

Missing Data

29 — 20 — 14 —

A. Unprocessed

97 — 66 — 43 —

Retained Data

29 — 20 — 14 —

B. Desalted

97 — 66 — 43 —

Missing Data

29 — 20 — 14 —

C. Precipitated

Figure 4. Comparison of unprocessed, desalted and precipitated nuclear extract. Nuclear extract (2 mg/ml) was prepared using the Thermo Scientific 2-D Sample Prep for Nuclear Proteins. Approximately 30 µg of protein was focused on a pH 4-7 IPG strip followed by 8-16% SDS-PAGE and the 2-D gels were silver-stained. A. unprocessed, B. desalted with the 2-D Sample Prep for Nuclear Proteins and C. precipitated with a commercial precipitation kit.

Ordering Information Product # Description 89863

2-D Sample Prep for Nuclear Proteins Kit Sufficient reagents for 25 applications. Includes: NE-PER Nuclear and Cytoplasmic Extraction Reagents: Cytoplasmic Extraction Reagent I (CER I) Cytoplasmic Extraction Reagent II (CER II) Nuclear Extraction Reagent (NER) 2-D Sample Buffer for Nuclear Proteins: 2-D Sample Buffer for Nuclear Proteins, Component A 2-D Sample Buffer for Nuclear Proteins, Component B Protein Desalting Spin Columns

89865

2-D Sample Prep for Soluble Proteins Kit Sufficient reagents for 25 applications. Includes: 2-D Sample Buffer for Soluble Proteins: 2-D Sample Buffer for Soluble Proteins, Component A 2-D Sample Buffer for Soluble Proteins, Component B Protein Desalting Columns

Pkg. Size Kit

5 ml 0.275 ml 2.5 ml

18 ml 16.5 g 25 columns

Kit 19.5 ml 15 g 25 columns

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

29

Thermo Scientific Products for Protein Detection and Gel Separation Table 2. 500 µl of the Thermo Scientific 2-D Marker Mix is sufficient for the following number of applications, depending on gel size and staining method.

2-D Protein Molecular Weight Marker Mix A 2-D gel marker mix with all the proteins you want – with no document to sign. Ready-to-use Thermo Scientific 2-D Protein Molecular Weight Gel Marker Mix was designed specifically to aid the proteome analyst. This 2-D gel marker mix contains a complement of seven reduced and denatured proteins. When performing protein 2-D separation and analysis, each protein in the mix provides important features for assessing system performance or estimating pI and molecular weight values. It’s also convenient – this marker mix doesn’t require you to sign a notarized document just to use it. A unique complement of proteins Each protein in this marker mix was carefully selected to give a useful range of molecular weight and pI coverage. Proteins were selected that give a variety of features from tight single spots to characteristic charge trains to aid the analyst in gel orientation. Molecular weights range from 17 K to 80 K, with pI values ranging from 4.5 to 8.7.

Gel Size

Stain Method

Marker Volume

# of Gels/ Vial

Mini-Gels

Coomassie Blue Dye

2.5 µl

200

Mini-Gels

Silver

0.5-1.0 µl

500-1,000

Large Format Gels

Coomassie Blue Dye

5-7.5 µl

66-100

Large Format Gels

Silver

1.0-2.5 µl

200-500

The 2-D Protein Molecular Weight Marker Mix is supplied frozen. For optimal long-term stability, aliquot into sample vials upon receipt and refreeze.

Ordering Information Product # Description 26659

Coomassie Blue Dye Stains 24590

GelCode Blue Stain Reagent

500 ml

Stains up to 25 mini-gels.

GelCode Blue Stain Reagent

3.5 L

Stains up to 175 mini-gels.

Silver Stain 24600

Pierce Silver Stain for Mass Spectrometry

Kit

Spot

2-D Marker Protein

MW

pI Value

A.

Apo-Transferrin (human plasma)

80K

6.2

Other Protein Molecular Weight Markers

B.

Glutamic Dehydrogenase (bovine liver)

56K

6.5, 6.7, 6.9

26691

Pierce 3-Color Prestained Protein Molecular Weight Marker Mix

C.

Actin (bovine muscle)

43K

5.2

1 x 48 microtube plate

D.

Carbonic Anhydrase (bovine erythrocytes)

29K

6.3

26681

E.

Myokinase (chicken muscle)

22.5K

8.7

Pierce Blue Prestained Protein Molecular Weight Marker Mix

1 x 48 microtube plate

F.

Trypsin Inhibitor (soybean)

20K

4.5

G.

Myoglobin (equine skeletal muscle)

17K

7.0, 7.4

A

A

B

B

C

C

F

A. Stained with Silver

E

D

E

D

F G

G

B. Coomassie Blue Dye

Figure 5. Thermo Scientific 2-D Marker shown stained with A. Silver and B. Coomassie Blue Dye.

30

500 µl

Compatible Products for use in Proteome Analysis

24592 Table 1. Component proteins in the Thermo Scientific 2-D Protein Molecular Weight Marker Mix.

2-D Protein Molecular Weight Marker Mix

Pkg. Size

For more information, or to download product instructions, visit www.thermo.com/pierce

Imperial Protein Stain MS can be effectively combined with staining for protein identification. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein separation, the individual protein bands are stained to detect low quantities of proteins adequate for downsteam MS analysis. We offer an exceptional line of stains for detecting proteins in gels. These stains not only offer superior performance for standard detection, but they are directly compatible with MS applications. Each stain produces little to no protein fixation or modification for clean and accurate mass spectral analysis. Thermo Scientific Imperial Protein Stain, a ready-to-use coomassie stain for detecting protein bands in SDS-polyacrylamide and 2-D gels, was designed for researchers who require maximum sensitivity and speed. The stain is a unique formulation of coomassie R-250 that delivers substantial improvements in protein-staining performance compared with homemade or other commercial stains (Figure 6). Multiple staining protocols are provided to meet demanding time and sensitivity requirements. Bands containing as low as 6 ng protein can be detected in as little as 20 minutes (Figure 8). Imperial Stain does not require a fixation step and uses a simple water wash to yield a clear background.

1. Wash the gel three times with deionized water (15 minutes).

Convenience • Destain with water • No fixation step required • Ready-to-use reagent • Stable – store on your bench top for up to one year • Flexible – multiple protocols to meet demanding time/sensitivity requirements

DI H2O

2. Add Imperial Protein Stain (5 minutes-1 hour).

3. Water destain (15 minutes-overnight).

Figure 7. Thermo Scientific Imperial Protein Stain protocol.

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

7

8

9

Rabbit IgG

BSA Protein A Protein G Lysozyme

5-minute stain; 15-minute water destain

1

Highlights: • Outstanding performance • Mass spectrometry-compatible • Sensitive – 3 ng protein/band and less can be detected with the enhanced protocol (3 hours) • Fast – detect down to 6 ng protein/band in just 20 minutes • Robust – highly consistent, reproducible protein staining • Excellent photo-documentation – photographs/scans better than other coomassie stains

S

DI H2O

2

3

4

5

6

7

8

1-hour stain; 2-hour water destain

9

Rabbit IgG

BSA Protein A Protein G Lysozyme

1-hour stain; overnight water destain

Figure 8. Enhanced sensitivity and crystal-clear background using Thermo Scientific Imperial Protein Stain. For even greater sensitivity and reduced background, gels are stained with Imperial Protein Stain for 1 hour and destained in water from 1 hour to overnight. Lane 1. BSA only (6 µg), Lanes 2-9 contained the indicated proteins at the following concentrations: Lane 2. 1,000 ng, Lane 3. 200 ng, Lane 4. 100 ng, Lane 5. 50 ng, Lane 6. 25 ng, Lane 7. 12 ng, Lane 8. 6 ng and Lane 9. 3 ng.

Ordering Information Product # Description

Pkg. Size

24615

Imperial Protein Stain

1L

24617

Imperial Protein Stain

3x1L

Figure 6. Thermo Scientific Imperial Protein Stain reveals spots that are faint or not detected with other Coomassie stains. Mitochondrial protein extract was prepared from heart tissue of six-week-old Sprague-Dawley rat. Processed protein extract (72 µg) was focused on a pH 5-8 IPG strip followed by 8-16% SDS-PAGE. The gels were stained for 1 hour and destained overnight following manufacturer-recommended protocols.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

31

Thermo Scientific Products for Protein Detection GelCode Blue Stain Reagent A sensitive 2-D electrophoresis (2-DE) stain that is mass spec-compatible. Highlights: • No destaining steps required for 2-DE – never needs pungentsmelling methanol/acetic acid solvents1 • Fast – works with a one-step, one-hour staining • More sensitive than standard coomassie gel stain formulations • Optional Water Wash Enhancement™ Step increases staining sensitivity • Easily washed away prior to MS analysis1,2 • Compatible with MALDI-TOF analysis3,4,5 • Compatible with sequence analysis6

References 1. Aulak, K.S., et al. (2001). Proteomics method identified proteins nitrated in vivo during inflammatory challenge. Proc. Natl. Acad. Sci. USA 98(21), 12056-12061. 2. Hilton, J.M., et al. (2001). Phosphorylation of a synaptic vesicle-associated protein by an inositol hexakisphosphate-regulated protein kinase. J. Biol. Chem. 279(19), 16341-16347. 3. Aulak, K.S., et al. (2001). Proc. Natl. Acad. Sci. USA 98, 12056-12061. 4. Lim, J., et al. (2002). J. Biol. Chem. 277, 20774-20782. 5. Hilton, J.M., et al. (2001). J. Biol. Chem. 276, 16341-16347. 6. Tani, M., et al. (2002). J. Biol. Chem. 275, 3462-3468.

Ordering Information Product # Description 24590

GelCode Blue Stain Reagent

Pkg. Size 500 ml

Sufficient for staining up to 25 SDS-PAGE mini gels (8 cm x 10 cm).

24592

GelCode Blue Stain Reagent*

3.5 L

Sufficient for staining up to 175 SDS-PAGE mini gels (8 cm x 10 cm).

72300

Pump (for 3.5 L package only)

1 pump

* A complimentary reagent dispensing pump attachment is available free upon request for Product # 24592. Specify Product # 72300 when you place your order.

Figure 1. A Thermo Scientific GelCode Blue Stain Reagent-stained 2-D gel. The gel is an isolated hydrophilic fraction obtained using the Thermo Scientific Mem-PER Kit (Product # 89826) from NIH 3T3 cells run on a pH 5-8 IPG strip. Approximately 80 µg of protein was applied to the gel. The second dimension was performed on an 8-16% SDS-PAGE.

32

For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Silver Stain for Mass Spectrometry

250 1440.5

200

1923.0 1601.8

Intensity

Silver stains offer exceptional detection characteristics, enabling detection of bands containing < 0.25 ng of protein. However, silver stain formulations are often incompatible with MS applications because of fixation and crosslinking that can occur during staining. We recognized the need for a silver stain that is not only compatible with MS applications, but is truly optimized to provide the best results. Thermo Scientific Pierce Silver Stain for Mass Spectrometry (Product # 24600) provides superior reliability, sensitivity and robustness for MS-target applications.

150 1989.0

100

1815.9 1385.8

569.4

50

677.2

1038.7 842.5 882.6 975.7 793.5 1090.0 1226.0

0 600

800

1,000

1,200

1,400

1,600

1,800

m/z

A. 250 1439.9 1923.0

Intensity

200

Figure 2. 2-D gel analysis of rat mitochondrial preparation (stained with Thermo Scientific Pierce Silver Stain for MS.) A preparation of rat mitochondria was extracted, loaded and separated using identical 2-D gel conditions. All gels were electrophoresed in a pH 5-8 gradient. Each gel was stained with Pierce Silver Stain for Mass Spectrometry (shown), Supplier I MS compatible stain and Thermo Scientific GelCode Blue Stain Reagent, respectively. Ten spots were identified that stained well for all staining conditions. These spots were picked for in-gel digestion and MS analysis. Proteins Identified 1 ATP synthase, H+ transporting, mitochondrial F1 complex, β-subunit

Methods All

2

Thermo Scientific Pierce Silver Stain for MS and Thermo Scientific GelCode Blue Stain Reagent Supplier I

3 4

5 6 7 8

9 10

AJ18 protein

ATP synthase, H+ transporting, mitochondrial F1 complex, subunit Δ Electron transfer flavo protein (ETF protein) H+ transporting two-sector ATPase (EC 3.6.3.14), α-chain precursor

Unknown protein for MGC:93808 Mitochondrial aldehyde dehydrogenase precursor Glutamate dehydrogenase 1 Glucose-regulated protein ER-60 protease Enoyl coenzyme A hydratase, short-chain mitochondrial

Translocase of inner mitochondrial membrane homolog 44 Enoyl coenzyme A hydratase, short-chain mitochondrial ATP synthase, H+ transporting, mitochondrial F1 complex, β-subunit

All Thermo Scientific Pierce Silver Stain for MS and Thermo Scientific GelCode Blue Stain Reagent Supplier I All All All Thermo Scientific Pierce Silver Stain for MS and Thermo Scientific GelCode Blue Stain Reagent Supplier I

150 1601.0

1989.0

100 1815.9

50

1038.7

569.4

842.6 679.6

0 600

800

1,000

1,200

1,400

1,600

1,800

m/z

B.

Figure 3. Comparative peptide fingerprint analysis of 2-D spot #1 by MS. MALDI ion trap MS was performed on an Agilent Technologies LC/MSD Trap XCT. Indicated spots (Figure 4) were picked from 2-D gels run identically with mitochondrial extract. Spots from both the A. Thermo Scientific Pierce Silver Stain for MS- and B. Thermo Scientific GelCode Blue Reagent-stained gels were reduced, alkylated and trypsinized using the Thermo Scientific In-Gel Tryptic Digestion Kit (Product # 89871). Peptide mass fingerprinting was performed with ProteinProspector. Both analyses identified the same mitochondrial protein: ATP Synthase, H+ Transporting, Mitochondrial F1 Complex.

Ordering Information Product # Description 24600

Pkg. Size

Pierce Silver Stain for Mass Spectrometry

Kit

Sufficient reagents to stain up to 20 SDS-PAGE mini-gels (8 cm x 8 cm) and to destain more than 500 gel plugs for subsequent analysis by MS. Includes: Pierce Silver Stain Sensitizer Pierce Silver Stain Pierce Silver Stain Developer Pierce Silver Stain Enhancer Silver Destain Reagent A Silver Destain Reagent B

2 ml 500 ml 500 ml 25 ml 4 ml 14 ml

All All

33

Thermo Scientific Products for In-Gel Digestion In-Gel Tryptic Digestion Kit and Pierce C-18 Spin Columns Highlights of the In-Gel Tryptic Digestion Kit: • Convenience – includes reagents for destaining of coomassie or fluorescent dye-stained proteins, reduction and alkylation of cystines, and tryptic digestion • Robustness – procedure and reagents produce reliable digestions and data generation across a wide range of conditions without requiring optimization • Accuracy – contains highly purified and modified MS-grade trypsin with no chymotryptic activity and minimal autolytic activity Highlights of the C-18 Spin Columns: • Efficient contaminant removal – significantly reduces signal suppression, improves signal-to-noise ratios and sequence coverage and minimizes the need to repeat experiments due to failed or poor spectra • Robustness – works with a wide variety of load volumes and concentrations; no need to reduce sample volume before application • Convenience – easier handling and no special equipment required for processing multiple samples compared to tip-driven systems that require single-sample processing • Sensitivity – special C-18 resin allows excellent recovery percentages, even at low sample loads (≤ 200 fmol) Sensitivity and accuracy for MS analysis is obtained only if proper sample preparation is performed. Two key sample preparation steps are the fragmentation of proteins into peptides and subsequent concentration and clean up of peptides. The In-Gel Tryptic Digestion Kit and C-18 Spin Columns are tailored to the needs of MS analysis.

The In-Gel Tryptic Digestion Kit is a complete set of reagents to perform approximately 150 digestions on colloidal coomassie or fluorescent dye-stained protein bands. The kit includes modified porcine trypsin, destaining buffers, reduction reagents, alkylation reagents and digestion buffers along with detailed and simple instructions. Each component and step was optimized and balanced to produce complete, accurate and clean digests using a variety of conditions for dependable MS analysis (Figure 1). Our C-18 Spin Columns concentrate peptide samples and remove contaminants common to biological systems, increasing the sensitivity, reliability and quality of MS analysis. Each spin column contains a porous C-18 reverse-phase resin with excellent binding and recovery characteristics for a wide range of peptide concentrations. The spin column format allows simultaneous processing of multiple samples (10-150 µl) in approximately 30 minutes without laborious, repetitive pipetting or specialized equipment (Figure 2). The columns effectively process peptides derived from ≤ 10 ng or up to 30 µg of protein. Sensitivity and detection limits are dependent on the downstream application. Methods Cytosolic and mitochondrial protein extracts were prepared from NIH 3T3 cells and separated by 2-D electrophoresis. Gels were stained with GelCode Blue Stain Reagent (Product # 24592) and excised using a manual spot picker. Protein bands were treated with the In-Gel Tryptic Digestion Kit and prepared for MALDI analysis with the C-18 Spin Columns. After processing the sample was dried in a vacuum evaporator for approximately 20 minutes, then resuspended in 1.1 µl of matrix (10 mg/ml α-cyano-4-hydroxycinnamic acid in 65% acetonitrile, 0.1% trifluroacetic acid). This mixture was spotted and analyzed by mass spectrometry.

2-D Gel

Excised protein bands. Add 200 µl Destaining Solution

1. Destain gel slices. (2 x 30 minutes at 37°C)

30 µl Reduce Buffer

30 µl Alkylation Buffer

2. Reduce. (10 minutes at 60°C)

200 µl Destaining Solution

3. Alkylate. 4. Wash. Perform in the dark (2 x 15 minutes at 37°C) (1 hour at room temperature)

Steps 2-4 are optional, but recommended.

Figure 1. Schematic protocol for the Thermo Scientific In-Gel Tryptic Digestion Kit.

34

For more information, or to download product instructions, visit www.thermo.com/pierce

Add 50 µl 100% ACN

5. Shrink and dry gel slice. (25 minutes)

35 µl Digestion Buffer and Activated Trypsin

6. Digest. (4 hours at 37°C or overnight at 30°C)

The In-Gel Tryptic Digestion Kit produced clean, high-sequence coverage digests containing minimal autolysis products. Our C-18 Spin Columns efficiently purified and concentrated digests for MALDI-TOF analysis. MS analysis of digests that were not concentrated with C-18 yielded little to no signal (data not shown).

Ordering Information

100

2242.1354

80

1425.6243

% Intensity

70 60

963 5254 1059 5423

50 40

1711.7308

842.5 00

30 20

1196.6809 1219.6092

10 0 700

1,060

1,420

1,780

2,140

2,500

Mass (m/z)

A.

2.6E+4

100

1553.7458

90 80 70 60

892.4963

50

1026.5919

40

723.4489

30

Product # Description

2.1E+4

1723.8519

90

% Intensity

Results and discussion The In-Gel Tryptic Digestion Kit and Pierce C-18 Spin Columns were used in the preparation of 2-D gel electrophoresis-separated proteins from mitochondrial isolates for MALDI-TOF analysis (see Figures 3, A-C for the mass spectra). Database searches identified unknown proteins as A) glutamate dehydrogenase, B) ATP synthase alpha chain and C) voltage-dependent anion selective channel protein 1, with 55.8, 53.3, and 46.0% sequence coverage, respectively.

1120.7355

1575.7776 1287 6963

20

Pkg. Size

2338.1694

10

89871

In-Gel Tryptic Digestion Kit

Kit

Sufficient for approximately 150 in-gel digestions. Includes: Modified Trypsin Trypsin Storage Solution Acetonitrile Ammonium Bicarbonate Tris[2-carboxyethyl]phosphine (TCEP) Iodoacetamide

20 µg 40 µl 70 µl 300 mg 500 µl 500 mg

0 700

1,060

1,420

1,780

2,140

2,500

Mass (m/z)

B. 100

842.5100 854.4695 917.2736

90 80

2.4E+4 1400.6435

Pierce C-18 Spin Columns Each column contains 8 mg of C-18 resin.

89873

Pierce C-18 Spin Columns Each column contains 8 mg of C-18 resin.

25 spin columns 50 spin columns

% Intensity

70

89870

60

2128.0591

50

2176.0994

40

1528.7888

30 20 1575.7776

10 0 700

1,060

1,420

1,780

2,140

2,500

Mass (m/z)

C.

Figure 3. MALDI-TOF MS of unknown proteins. Samples were processed with the Thermo Scientific In-Gel Tryptic Digestion Kit and Pierce C-18 Spin Columns.

1. Activate Resin

2. Equilibrate Resin

3. Bind Sample

4. Wash

5. Elute

6. Dry and resuspend for MS Analysis

Wet resin with 200 µl 50% methanol or 50% ACN. Centrifuge and repeat.

Equilibrate resin with 200 µl 5% ACN, 0.5% TFA. Centrifuge and repeat.

Load 10-150 µl of sample to wetted resin. Centrifuge and repeat.

3-5 minutes

3-5 minutes

3-5 minutes

Wash resin with 200 µl 0.5% TFA in 5% ACN. Centrifuge and repeat 1-2 additional times.

Elute sample using 20 µl 70% ACN. Centrifuge and repeat. 3 minutes

5 minutes

Figure 2. Schematic protocol for Thermo Scientific Pierce C-18 Spin Columns.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

35

Thermo Scientific Products for In-Solution Digestion

In-Solution Tryptic Digestion and Guanidination Kit Analyze proteins by mass spec with confidence. Highlights: • Optimized – complete digestion is achieved for 0.025-10 µg protein samples with minimal to no side reactions • Quick – protein can be reduced, alkylated, digested and guanidinated all in one day • Convenient – kit includes reagents for reduction, alkylation, digestion and guanidination Trypsin specifically cleaves peptide bonds at the carboxyl side of arginine and lysine residues, generating a peptide map unique for each protein. Analysis of tryptic peptides by mass spectrometry (MS) provides a powerful tool for identifying proteins or analyzing post-translational modifications. Reliable mass spectral analysis requires accurate and complete digestion of the target proteins as well as modification of peptides to optimize ionization and detection. The Thermo Scientific In-Solution Tryptic Digestion and Guanidination Kit contains optimized procedures and reagents for reduction, alkylation, digestion and guanidination to provide reliable MS analysis of approximately 90 protein samples containing 0.025-10 µg of protein (Figure 1). The In-Solution Tryptic Digestion and Guanidination Kit contains a proteomics-grade modified trypsin that produces clean, complete digests with minimal autolysis products present. A reduction and alkylation protocol eliminates disulfide bonds, improving peptide identification and simplifying data analysis. Guanidination eliminates ionization bias between peptides with C-terminal arginine residues over C-terminal lysine residues, improving detection and overall sequence coverage.

36

In the example below, a variety of proteins (200 ng per sample) were processed using the In-Solution Tryptic Digestion and Guanidination Kit. Following reduction, alkylation and digestion, each sample was divided in half. One-half was guanidinated, and the other half was saved as a no-guanidination control. Each sample was processed by Pierce C-18 Spin Columns and then analyzed on an Agilent LC/MSD Trap XCT equipped with an APMALDI ionization source using α-CHCA as the matrix. Proteins processed with the In-Solution Tryptic Digestion and Guanidination Kit produced clean and reliable mass spectra with high sequence coverage (Table 1, Figure 2). Using the guanidination procedure to convert lysines to homoarginines enhanced the overall signal intensity of lysine-containing peptides by an average of 1.5- to 4-times, eliminating the ionization bias for peptides with a terminal arginine and improving sequence coverage and the reliability of data analysis. Table 1. Sequence coverage data for tryptic digestions with and without guanidination for three proteins. Sequence Coverage Protein

No Guanidination

With Guanidination**

Lysozyme (14,000 MW)

6/8 peptides 66/86aa 77%

8/8 peptides 86/86aa 100%

Myoglobin (17,000 MW)

6/12 peptides 78/134aa 58%

8/12 peptides 90/134aa 67%

BSA (66,000 MW)

25/44 peptides 318/489aa 65%

28/44 peptides 344/489aa 70%

** High levels of sequence coverage occur for all test proteins processed with the Thermo Scientific In-Solution Tryptic Digestion and Guanidination Kit with significant increase in sequence coverage for samples undergoing the guanidination procedure. Sequence coverage based only on those peptides expected to be identified based on scanning from 600-2,000 m/z.

For more information, or to download product instructions, visit www.thermo.com/pierce

5

Guanidinate 1

2

Denature/ Reduce 5 minutes at 95°C

3

Alkylate

4

12 minutes at 65°C

6

Digest

5

Process for MS Analysis

2 hours at 37°C or overnight at 30°C

Split for Non-Guanidinated Control (Optional)

Digest

20 minutes at room temp

3 hours at 37°C

Figure 1. Thermo Scientific In-Solution Tryptic Digestion and Guanidination Kit protocol.

Intens. x104

Intens. x104 1.5

1.5

1.0

1.0

0.5

0.5

# 0.0

600

700

800

900

*

1000

1100

0.0

A.

(m/z)

0.0

A. 1200

1300

1400

1500

1600

1700

1800

1900

0.0

(m/z)

0.5

0.5

# 1.0

1.0

1.5

1.5

B.

Figure 2. Comparison of a guanidinated digest with a non-guanidinated control demonstrates improved ionization and sequence coverage after guanidination. A. MS spectra of digested BSA (100 ng). B. MS spectra of digested BSA (100 ng) with guanidination. Guanidination results in a mass shift of +42 m/z for each lysine in a peptide. Symbols (#) and (*) indicated two lysine-containing peptides with and without guanidination for demonstration purposes.

*

B.

Ordering Information Product # Description 89895

In-Solution Tryptic Digestion and Guanidination Kit Sufficient reagents for approximately 90 digests of samples containing 0.025-10 µg of protein. Includes: Modified Trypsin Trypsin Storage Solution Ammonium Bicarbonate No-Weigh DTT Iodoacetamide (IAA) O-Methylisourea Hemisulfate Salt (OMI) Ammonium Hydroxide

Pkg. Size Kit

20 µg 50 µg 50 mg 7.7 mg 500 mg 400 mg 1 ml

Complementary Products 28904

TFA, Sequanal Grade (10X)

10 x 1 ml

89870

Pierce C-18 Spin Columns

25/pkg.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

37

Thermo Scientific Products for Peptide Enrichment

A novel tool for isolating phosphorylated peptides from complex protein digests. The central role of reversible protein phosphorylation/dephosphorylation in regulating cell function makes protein phosphorylation a major topic of study and one of the cornerstones of proteomic research. Identifying phosphoproteins and their phosphorylation sites is a difficult endeavor, requiring a variety of techniques. The Thermo Scientific Phosphopeptide Isolation Kit (Product # 89853) simplifies the isolation and identification of phosphopeptides from protein digests through the specific interaction of phosphate groups with immobilized gallium. This kit has a high binding capacity for phosphopeptides (~150 µg), making it compatible with classical methods for phosphopeptide analysis (e.g., 32P radiolabeling, Edman degradation and TLC/HPLC), yet it is more sensitive and more powerful for LC-MS and MALDI-TOF mass spectrometric (MS) methods because samples are eluted directly in MS-compatible buffers.

• Ideal for MS analysis – simplifies MS analysis by isolating phosphorylated peptides from complex mixtures; peptides can be eluted with MS-compatible buffers • Reliable – isolates single and multiple phosphorylated peptides; low nonspecific binding • Room temperature stable – one year shelf life from the date of purchase

Relative Intensity

Phosphopeptide Isolation Kit

2.3E+4

100 830 90 80 70 60 780 50 646 40 742 30 748 20 10 0 500

2,910

2,186

1,200

1,900

2,600

3,300

4,000

Mass (m/z)

The Phosphopeptide Isolation Kit uses a gallium-chelated IDAbased resin that provides less nonspecific peptide binding and greater specificity for both single and multi-phosphorylated peptides than iron-chelated and NTA-based resins.2,3,4 Additionally phosphopeptide isolation can be performed using MS-compatible elution buffers such as 0.1 N ammonium hydroxide or 100 mM ammonium bicarbonate, allowing direct analysis of sample with no desalting or additional sample handling required.1,2,3 The MALDI-TOF mass spectrum of peptides isolated from the tryptic-digested casein sample is shown in Figure 2. Peaks at masses 2,062 and 3,123 are the main signals detected in the eluted sample and represent peptides of 1,981 Da containing one phosphate group and of 2,802 Da containing four phosphate groups (+80 Da for each phosphate). Highlights: • Fast – total procedure time is just 15 minutes • Easy to use – no metal chelating or resin equilibration required; each mini spin tube contains one ready-to-use SwellGel Gallium Disc • High binding capacity – each SwellGel Gallium Disc is made of 25 µl gallium (III) resin with a binding capacity for phosphopeptides of approximately 150 µg

38

Figure 1. MALDI-TOF mass spectrum of tryptic digest of β-Casein. Peaks at masses 646, 742, 748, 780, 830 and 2,186 represent the expected peptides from a tryptic digest of β-casein. Peaks for the known phosphopeptides at 2,062 Da and 3,123 Da are not resolved in positive ion mode due to the additional negative charge. The peak at mass 2,910 represents a partial digest fragment. 2.4E+4

2,062

1,200

1,900

3,025

2,795

2,557

1,963 1,971

1,529

1,360

830

971 1,013 1,071

568

3,123

1,516 1,660

100 90 80 70 60 50 40 30 20 10 0 500

Relative Intensity

Beta-Casein was digested with immobilized trypsin for 4 hours at 37°C and 3 µl (approximately 5 µg) of this digest was adjusted to pH 3.0 by adding 50 µl 0.1% acetic acid. The sample was applied to the column and washed with 75 µl of 0.1% acetic acid, followed by 75 µl of 0.1% acetic acid, 10% acetonitrile and a final wash of 75 µl water. Sample was then eluted in three separate fractions using 20 µl of 100 mM ammonium bicarbonate. Elution # 1 (0.5 µl) was mixed with 0.5 µl of MALDI matrix (saturated α-cyano-4hydroxycinnamic acid in 50% acetonitrile, 0.1% trifluoroacetic acid) and 0.5 µl ammonium citrate to improve detection of phosphopeptides. This mixture was spotted and analyzed in positive ion, linear, delayed-extraction mode on an Applied Biosystems Voyager DE™ PRO MALDI-TOF mass spectrometer (Figures 1 and 2).

2,600

3,300

4,000

Mass (m/z)

Figure 2. MALDI-TOF mass spectrum of tryptic digest of β-Casein after sample was processed with the Thermo Scientific Phosphopeptide Isolation Kit. The 2,062 peak is the single phosphorylated peptide representing the amino acid sequence from residue 48 to 63. The 3,123 peak is the peptide from residue 16 to 40 containing four phosphate groups. None of the non-phosphorylated peptides of β-casein are detected in significant quantities. References 1. Giorgianni, F., et al. (2004). Identification and characterization of phosphorylated proteins in the human pituitary. Proteomics 4, 587-598. 2. Poseqitz, M.C. and Tempst, P. (1999). Immobilized gallium (III) affinity chromatography of phosphopeptides. Anal. Chem. 71, 2883-2892. 3. Zhou, W., et al. (2000). Detection and sequencing of phosphopeptides affinity-bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry. J. Am. Soc. Mass. Spectrom. 11, 273-282. 4. Ficarro, S.B., et al. (2002). Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nature Biotechnology 20, 301-305.

Ordering Information Product # Description 89853

Phosphopeptide Isolation Kit Sufficient discs to analyze 30 protein digestions. Kit contains: 30 mini-spin columns containing a SwellGel Gallium (III) Disc.

For more information, or to download product instructions, visit www.thermo.com/pierce

Pkg. Size Kit

Thermo Scientific Products for Peptide Clean-up Pierce Strong Cation and Anion Ion Exchange Spin Columns Charge through your purifications with ease. Ion exchange chromatography separates molecules based on differences in their accessible surface charges. This technique is widely used in the pre-fractionation or purification of a target protein from crude biological samples. Novel membrane-based ion exchange chromatography is attracting attention because of its advantages over resin-based column chromatography. Membranebased ion exchange chromatography has shorter diffusion times than resin-based chromatography. Interactions between molecules and active sites on the membrane occur in a convective manner through pores, which shortens the diffusion time compared with fluid inside the pores of a resin particle. Also, the relatively mild binding and eluting conditions of this separation method produce high protein recovery with intact biological activity. Thermo Scientific Pierce Strong Cation and Anion Ion Exchange Spin Columns use the membrane-adsorber technology as a chromatographic matrix to fractionate proteins based on their charge differences. The membrane adsorbers have a highly porous structure with pores larger than 3,000 nm, providing proteins easy access to the membrane’s charged ligands. Therefore, adsorptive membranes maintain high efficiencies at high-flow rates and when fractionating large biomolecules with small diffusivities. Highlights: • Fast and simple – membrane-based spin format eliminates column packing • Convenient and expandable – centrifugal format enables convenient processing of multiple samples in parallel • Robust – membrane adsorber spin columns do not crack or run dry • Low bed volume – small membrane adsorber bed volumes make working with low buffer volumes possible, leading to concentrated elution fractions Our Strong Cation and Anion Ion Exchange Spin Columns are available in two sizes: • Mini, offering binding capacities of 4 mg (500 µl) • Maxi, offering binding capacities of 80 mg (20 ml)

Applications: • Purification of phosphopeptides before MS analysis • Pre-fractionation of proteins in lysate • Scouting purification conditions for new protein preparation protocols • Removal of endotoxins from monoclonal antibodies • Polishing His-tagged proteins after metal chelate chromatography • Purification and concentration of proteins and viral particles • Purification of antibodies from serum, ascites or tissue culture supernatant • Removal of detergent from protein solutions • Sample preparation before 1D or 2D PAGE Figure 1. Procedure summary.

Ordering Information Product # Description

Pkg. Size

SPIN

Original sample

SPIN

Protein is bound to membrane

Wash

Elute

Pure protein

90008

Strong Cation Exchange Spin Column, Mini

24 Spin Columns

90009

Strong Cation Exchange Spin Column, Maxi

8 Spin Columns

90010

Strong Anion Exchange Spin Column, Mini

24 Spin Columns

90011

Strong Anion Exchange Spin Column, Maxi

8 Spin Columns

Complementary Products 89853

Phosphopeptide Isolation Kit

Kit

Sufficient for analyzing 30 protein digestions. Kit contains: Mini-spin columns each contain one SwellGel Gallium-Chelated Disc

30 ea.

Acknowledgments: We would like to thank Dr. Bassam Wakim, Michael Pereckas and Dr. Joyce Thompson at the Protein and Nucleic Acid Core Facility at the Medical College of Wisconsin, Milwaukee, for their helpful contribution with mass spectrographic analysis of phosphopeptides.

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

39

Thermo Scientific Products for Mass Spectrometry Ion Pairing Reagents Ion pairing agents are essential for studying proteins and peptides by LC-MS and MALDI-MS. For complex peptide separations and analysis, the ion pairing reagent is often the key to success. Varying mobile phase composition on the same separation column or sample handling device can often change selectivity enough to resolve overlapping peaks or isolate previously unidentified peptides. We offer the commonly used ion pairing reagents, trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA), in exceptional purity (> 99.5%) and clarity and high-purity Formic Acid in 1 ml ampules to simplify preparation of HPLC mobile phases. TFA is the most commonly used ion pairing agent in reverse-phase peptide separations because it:

Ordering Information Product # Description 28901

Trifluoroacetic Acid, Sequanal Grade

500 ml

28902

Trifluoroacetic Acid (TFA), Sequanal Grades for making 0.1% w/v TFA solutions

10 x 1 g

28903

Trifluoroacetic Acid, Sequanal Grade

100 g

28904

Trifluoroacetic Acid (TFA), Sequanal Grade for making 0.1% v/v TFA solutions

10 x 1 ml

25003

Heptafluorobutyric Acid

100 ml

53104

Heptafluorobutyric Acid

10 x 1 ml

28905

Formic Acid, 99+%

10 x 1 ml

Crosslinking Technical Handbook

• Sharpens peaks and improves resolution • Is volatile and easily removed • Has a low absorbance within the wavelengths of detection • Is a proven reagent for peptide and protein analysis

Request your free copy of the Crosslinking Technical Handbook today! ®

Thermo Sc entific Pierce Crossl nking Technical Handbook

Highlights: • Excellent purity – ≥ 99.5% pure with exceptional clarity, allowing sensitive, nondestructive peptide detection at low UV wavelengths in reverse-phase HPLC protein and peptide separation systems • Integrity – packaged under nitrogen in amber glass ampules or bottles with protective TFE-lined fluorocarbon caps for high performance

40

Pkg. Size

For more information, or to download product instructions, visit www.thermo.com/pierce

Our Crosslinking Technical Handbook (Product # 160167 ) provides an introduction to crosslinking, an overview of applications and chemistries, a selection guide, and other technical and product information to help maximize results for crosslinking procedures. To request a free copy or to view an online selection guide, visit our website.

Mass Spectrometry Applications Crosslinking Reagents for the Analysis of Protein Interactions by Mass Spectrometry Analysis Homobifunctional Crosslinking Reagents for MS Analysis As the proteome becomes more defined, protein interactions become increasingly important to understand. Protein modification, labeling and Thermo Scientific Pierce Crosslinking Reagents have had a central role in the study of protein interaction for many years. The combination of MS and protein crosslinking further facilitates the studies of interactions.1-2

References 1. Sinz, A. (2003). J. Mass Spectrom. 38, 1225-1237. 2. Dihazi, G.H. and Sinz, A. (2003). Rapid Commun. Mass Spectrom. 17, 2005-2014. 3. Muller, D.R., et al. (2001). Anal. Chem. 73, 1927-1934. 4. Chen, X., et al. (1999). Anal. Biochem. 273, 192-203. 5. Bennett, K.L., et al. (2000). Protein Sci. 9, 1503-1518. 6. Back, J. W., et al. (2002). Protein Sci. 11, 2471-2478. 7. Wine, R.N., et al. (2002). Anal. Chem. 74, 1939-1945. 8. Itoh, Y., et al. (2001). Proc. Natl. Acad. Sci. USA 98, 4883-4887. 9. Pearson, K.M., et al. (2002). Rapid Commun. Mass Spectrom. 16, 149-159. 10. Schutz, D.M., et al. (2004). Biochemistry 43, 4703-4715.

MS analysis of crosslinking reactions can yield low-resolution three-dimensional protein structure information, providing insight into how a protein folds or helping identify sites and sequences responsible for protein interactions. DSG, BS3, Sulfo-EGS, DTSSP, Sulfo-SBED and BS2G are popular crosslinking reagents for use in MS analysis. To simplify the interpretation of resulting mass spectra from inter- or intramolecular crosslinking experiments, BS3 and BS2G are also available with defined isotope labeling (4 deuterium atoms). See our website for more information on our Crosslinking Reagents.

Ordering Information Product # Description 21580

Key Features

BS3

1,2,3,10

50 mg

MW 204.09 Spacer Arm 3.0 Å

• Amine reactive • Short distance cross-links

4

1g

MW 245.15 Spacer Arm 8.6 Å

• Amine reactive • Retains charge character

2.9

50 mg

MW 273.20 Spacer Arm 11.0 Å

• Amine reactive • Retains charge character

2

1g

MW 326.26 Spacer Arm 7.7 Å

• Amine reactive • Non-cleavable

1,3

50 mg

MW 608.51 Spacer Arm 12 Å

• Amine reactive • Reducing agents cleavable • Water soluble

5,6

50 mg

MW 548.32 Spacer Arm 6.4 Å

• Water soluble • Periodate cleavable • Protein S-S bonds remain intact

1,2

50 mg

MW 660.45 Spacer Arm 16.1 Å

• Water soluble • Hydroxyl-amine cleavable

1,2

50 mg

MW 621.60 Spacer Arm 23.6 Å

• Amine reactive • Photo-reactive • Fluorescent

7

5 mg

MW 312.37 Spacer Arm 6.8 Å

• Amine reactive • Thiol reactive • Cleavable by reducing agents

8

50 mg

N-Succinimidyl 3-(2-pyridyldithio)propionate

EDC

MW 191.70

• Cross-links –COOH with –NH2 • Amide linkage • Zero-length linkage

1

5 mg

DFDNB 1,5 Difluoro-2,4-dinitrobenzene)

20663

DMA Dimethyl adipimidate•2HCl

20700

DMS Dimethyl suberimidate•2HCl

20593

DSG Disuccinimidyl glutarate

21578

DTSSP 3,3’Dithiobis(sulfosuccinimidyl propionate)

20589

DST Disuccinimidyl tartarate

21566

Sulfo-EGS Ethylene glycol bis(sulfosuccinimidyl succinate)

33030

SAED Sulfosuccinimimidyl 2-(7-azido-4-methyl-coumarin-3acetamido)ethyl-1,3’dithio-propionate

21857

22980

Pkg. Size

• Water soluble • Amine reactive • Non-cleavable

Bis(Sulfosuccinimidyl) suberate

21525

Ref.

MW 572.43 Spacer Arm 11.4 Å

SPDP

1-Ethyl-3-(3-dimethyl-aminopropyl)carbodiimide HCl

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

41

Mass Spectrometry Applications Gain new insights into protein interaction and folding

O

Deuterated crosslinkers and mass spec: a powerful combination in protein structure-function analysis A rapidly emerging strategy to analyze protein structure and function integrates the proven usefulness of protein crosslinking reagents with the power of mass spectrometry (MS) to yield insights into protein tertiary1,6-8,10 structure and protein complex formation.1-6,11 We continue being a leader in protein crosslinking by synthesizing and characterizing the deuterated analogs of BS3 and BS2G (Figure 1) for the mass spectral study of intramolecular distances and intermolecular interactions.

O

Na+O–

D

O

S

N

O

Strategy for crosslinker reagent pairs and MS analysis Heavy (–dn) and light (–d0) crosslinker analogs are reacted simultaneously with the target protein or protein complex. Protein crosslinking and labeling occur in a single step. Using a 1:1 ratio of two identical crosslinking agents differing only in the number of deuterium atoms (e.g., d4 vs. d0) is a powerful identifier of low-abundant crosslinked peptides. MS patterns of the resultant crosslinked peptides differ by four mass units after enzymatic digestion (Figure 2). Further analysis of the reaction products can yield low-resolution, three-dimensional structure information. Intermolecular crosslinking of an interacting protein complex and MS analysis have been used to determine the molecular contact surfaces of binding partners in a protein complex.2,5

42

O O

O

O

D

S

O – O Na+

O

BS3-d4 MW 576.45 Spacer Arm 11.4 Å O

Na+O– O

O N

O

D

O

O

S

N

O

O

O

O

Highlights: • Well-characterized, high-purity, deuterium-labeled crosslinkers and their hydrogen-containing analogs • Instructions edited by an expert in the crosslinking/MS strategy field • Suberate- and glutarate-based reagent pairs – provide a “molecular ruler” option to study inter- and intra-molecular distances to gain structural information not possible with just one reagent • Efficient and convenient – requires only microgram amounts of protein; an excellent alternative to NMR or X-ray crystallographybased methods that require large amounts of protein, special solvents or crystal formation

D

S

O – O Na+

O

BS3-d0 MW 572.43 Spacer Arm 11.4 Å

O

O–Na+

O

O N

Na+O–

S

O

O

O

O

O

O

N

O

O O

O S

D

D

D

N O

D

BS2G-d4 MW 534.38 Spacer Arm 7.7 Å O–Na+

O

Na+O–

S

O

O

O

O N O

O

O O

O BS2G-d0 MW 530.35 Spacer Arm 7.7 Å

O S

N O

Figure 1. Structures of the heavy and light analogs of BS3 and BS2G developed for MS applications. Homobifunctional, amine-reactive, non-cleavable, watersoluble crosslinking agents with defined spacer arm lengths act as molecular rulers for estimation of spatial relationships in protein structure-function studies. Both the light analogs (BS2G-d0 and BS3-d0) and the heavy analogs (BS2G-d4 and BS3-d4) react efficiently with primary amino groups (–NH2) at pH 7-9 to form stable amide bonds. Proteins contain primary amines in the side chain of lysine (K) residues and the N-terminus of each polypeptide. All reagents are supplied as a sodium salt and are water-soluble up to 10 mM.

For more information, or to download product instructions, visit www.thermo.com/pierce

What is a heavy crosslinker? A heavy crosslinker is the deuterated analog of a specific crosslinking reagent. In a heavy crosslinker, deuterium (D) is substituted for hydrogen (H) on specific methylene carbons within the crosslinker hydrocarbon spacer arm. For reagents introduced here, –CH2- is converted to a –CD2- on two methylene carbons in their respective hydrocarbon spacer arms. Why are heavy crosslinkers key to structure-function analysis by MS? Peptide masses differing by four units unambiguously identify a crosslink at a specific locus within a protein structure or between protein-binding partners. Crosslinker spacer arm length can be used to assign distance constraints between crosslinked peptides. 100 2730.329 2726.307

R elative Intensity ( % )

75 2727

2731

2735 m/z

50

25 n.a

n.a.

n.a.

n.a. 0

1000

References Applications of Heavy/Light Crosslinking Reagents and Mass Spectrometry 1. Sinz, A. (2006). Chemical crosslinking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. Mass Spectrom. Rev. DOI:10.1002/mas.20082. 2. Schmidt, A., et al. (2005). Mapping protein interfaces by chemical cross-linking and FTICR mass spectrometry: Application to a calmodulin/adenylyl cyclase 8 peptide complex. Eur. J. Mass Spectrom. 11, 525-534. 3. Schmidt, A., et al. (2005). Studying calmodulin/adenylyl cyclase 8 interaction using isotope-labeled cross-linkers and FTICR mass spectrometry. Amer. Soc. Mass Spec. 53rd Conference (San Antonio, TX). Poster #361. See www.piercenet.com. Search on deuterated crosslinkers and click on Application 2. 4. Kalkhof, S., et al. (2005). Probing laminin self-interaction using isotope-labeled crosslinkers and ESI-FTICR Mass Spectrometry. Amer. Soc. Mass Spec. 53rd Conference (San Antonio, TX). Poster #352. See www.piercenet.com. Search on deuterated crosslinkers and click on Application 2. 5. Kalkhof, S., et al. (2005). Chemical cross-linking and high-performance fourier transform ion cyclotron resonance mass spectrometry for protein interaction analysis: Application to a calmodulin/target peptide complex. Anal. Chem. 77, 495-503. 6. Sinz, A. (2003). Chemical cross-linking and mass spectrometry for mapping threedimensional structures of proteins and protein complexes. J. Mass Spectrom. 38, 1225-1237. 7. Dihazi, G.H. and Sinz, A. (2003). Mapping low-resolution three-dimensional protein structure using chemical cross-linking and Fourier transform ion-cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2005-2014. 8. Back, J.W., et al. (2003). Chemical cross-linking and mass spectrometry for protein structural modeling. J. Mol. Biol. 331, 303-313. 9. Schilling, B., et al. (2003). MS2 assign, automated assignment and nomenclature of tandem mass spectra of chemically cross-linked peptides. J. Am. Soc. Mass Spectrom. 14, 834-850. 10. Pearson, K.M., et al. (2002). Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method. Rapid Commun. Mass Spectrom. 16, 149-159. 11. Muller, D.R., et al. (2001). Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis. Anal. Chem. 73, 1927-1934 12. Peri, S., et al. (2001). GPMAW – a software tool for analyzing proteins and peptides. Trends Biochem. Sci. 26, 687-689.

2000

3000

n.a. 4000

Ordering Information m/z

Figure 2. Deconvoluted ESI-FTICR mass spectrum of the tryptic peptide mixture from intramolecularly crosslinked calmodulin (CaM). One hundred-fold excess of BS2G-d0/d4 over protein/peptide concentration was used and incubated for 60 minutes. Legend: circle: CaM peptides, diamond: autolytic peptides from trypsin, star: crosslinker containing species. The insert is a crosslinking product within CaM sequence 14-37 in which Lys-21 has reacted with Lys-31. The 1:1 pattern with a mass difference of 4 u caused by the isotope-labeled crosslinker enhances confidence in the assignment of crosslinking products.2,9,12

For a schematic of analytical strategies for 3-D structure and protein interaction mapping, visit www.thermo.com/pierce. Once there, search on the product # for a deuterated crosslinker and then select General Protocol for Tertiary Structure and Protein Interaction Applications.

Product # Description 21590

Pkg. Size

BS3-d0

10 mg

BS3-d4

10 mg

BS2G-d0

10 mg

BS2G-d4

10 mg

Bis(sulfosuccinimidyl) suberate-d0

21595

Bis(sulfosuccinimidyl) 2,2,7,7 suberate-d4

21610

Bis(sulfosuccinimidyl) glutarate-d0

21615

Bis(sulfosuccinimidyl) 2,2,4,4 glutarate-d4

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

43

Thermo Scientific Products for Protein Quantification by Mass Spectrometry TMT Isobaric Mass Tagging Kits Sample 1

Label TMT6-126

Sample 2

Label TMT6-127

Sample 3 Sample 4 Sample 5

ne Combine

Label TMT6-130

Fractionate, Clean-up, LC-MS/MS analysis

Label TMT6-131

LC Separation p

m/z

Relative Abundance

MS Selection

m/z

O N O

Cleavable Linker

Label TMT6-129

Protein Reactive Group

Figure 1. Structural design of a tandem mass tag. Mass reporter: Each member has a unique mass and reports sample-specific abundance of a labeled peptide during MS/MS analysis. Cleavable linker: Preferentially fragments under typical MS/MS conditions to release the mass reporter. Mass normalizer: Each member has a unique mass that balances the mass reporter, ensuring the same overall mass for all tags in a set. Reactive group: Reactive NHS ester provides high-efficiency amine-specific labeling of proteins/peptides.

MS/MS Identification

MS/MS Quantitation

Intensity (Counts)

O

O

Mass Reporter Mass Normalizer

Label TMT6-128

O

N H

Denature, Reduce, Alkylate & Tryptic Digest

Sample 6

Thermo Scientific Tandem Mass Tags (TMT) are isobaric tags uniquely designed to enable a rapid and cost-effective transition from method development to high-throughput protein quantitation. The tags consist of TMT0 (zero), the TMT2 two-plex set and the TMT6 six-plex set. The TMT0 tag allows testing and optimization of sample preparation, labeling, fractionation and MS fragmentation for peptide identification and reporter detection without using the more costly isotope-labeled compounds. The TMT2 reagent set allows two-plex protein profiling for small studies. The TMT6 reagent set allows six-plex protein profiling for multiple conditions, including time courses, dose responses, replicates or multiple sample comparisons. Each TMT tag is based on the same chemical structure, eliminating the need to modify labeling conditions or HPLC separation conditions between experiments.

N

Treat Samples & Isolate Proteins

Intensity [counts] (10 9)

Isobaric chemical tags are powerful tools that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. Isobaric tags are small chemical molecules with identical structure that covalently bind to the free amino termini of lysine residues of peptides, thereby labeling various peptides in a given sample (Figure 2). During the MS/MS analysis, each isobaric tag produces a unique reporter ion signature that makes quantitation possible. In the first MS analysis, the labeled peptides are indistinguishable from each other; however, in the tandem MS mode during which peptides are isolated and fragmented, each tag generates a unique reporter ion. Protein quantitation is then accomplished by comparing the intensities of the six reporter ions in the MS/MS spectra.

m/z m/z

Descript on Isopentenyl diphosphate Delta isomerase 1 Isoform 1 of Growth factor receptor bound prote n 2 Hypothetical prote n U2 small nuclear ribonucleop otein A 14 3 3 protein sigma S gnal recogn tion particle receptor subunit beta Calcyclin b nding protein Vesicle assoc ated membrane p otein associated protein A Protein tyrosine k nase (Fragmen ) Isoform 1 of Estradiol 17 beta dehydrogenase 12 Hypoxanthine guanine phosphoribosyltransferase Proteasome subunit alpha type 1 Translin Enoyl CoA hydratase m tochondrial precursor Isoform Long of Spl c ng factor arginine/ser ne rich 3 Lactoylglutathione lyase Succinate dehydrogenase (ubiqu none) iron sulfur subunit mitochondrial precursor

Figure 2. Protein profiling with tandem mass tags. Proteins from up to six treated samples are: 1. denatured; 2. digested with trypsin; 3. labeled with TMT6 tags; 4. combined; 5. cleaned or fractionated by strong cation exchange; 6. chromatographically separated, isolated and fragmented as peptides by in-line reversed phase LC-MS/MS; and 7. identified and quantified with Thermo Scientific BioWorks, Discoverer or Matrix Science Mascot™ Search Engine.

44

For more information, or to download product instructions, visit www.thermo.com/pierce

Highlights: • Enabling protein ID and quantitation from multiple samples of cells, tissues or biological fluids • Consistent chemistry allowing efficient transition from method development to multiplex quantitation, enabling biomarker discovery research • Efficient labeling of membrane and post-transitionally modified proteins • Expandable system allows concurrent multiplexing of up to six different samples in a single experiment (Figure 2) • Optimized fragmentation and fully supported quantitation with Protein Discoverer™ 1.0 for all Thermo Scientific LC MS/MS platforms, such as LTQ™ XL and LTQ Orbitrap™ XL Systems • TMT0 allows testing and method optimization without more costly isotope-labeled compounds The TMT tags are provided as standalone sets or in optimized kit formats containing all necessary reagents and controls for maximum flexibility, convenience and reliability. The TMT Reagents combined with Thermo Scientific instruments and software provide a complete and integrated solution to perform absolute quantitation of target proteins (Figure 2).

Ordering Information Product # Description

Custom HeavyPeptides™ BASIC and AQUA Kits Custom kits for relative and absolute quantification of protein by LC-MS. Highlights: • Quantification of low abundance proteins • Higher accuracy, sensitivity and specificity • Easy to order, custom-made HeavyPeptides • Relative or absolute quantification • Economically viable for high throughput screening One of the key challenges in Proteomics is the quantification of proteins at very low concentrations in complex protein mixtures. The Thermo Scientific HeavyPeptide solution is based on the well established isotopic dilution technique. HeavyPeptide kits are the ultimate choice for specific, accurate and sensitive protein quantification. As one of the leading manufacturers of mass spectrometers we have a large base of customers confronted daily to these challenges. We were also the first company to install a pilot research center dedicated to Biomarker discovery [(BRIMS1), in Boston, MA. Carefully listening to these end-users led us to the development of a fast, economical, and easy-to-use solution: Custom HeavyPeptide Basic and AQUA2 kits.

Pkg. Size

90063

TMTduplex™ Isobaric Mass Tagging Kit Labeling Reagents for Multiplexed and Absolute Protein Quantification

Kit

90064

TMTsixplex™ Isobaric Mass Tagging Kit Labeling Reagents for Multiplexed and Absolute Protein Quantification

Kit

90065

TMTsixplex Label Reagent Set Labeling Reagents for Multiplexed and Absolute Protein Quantification

Kit

90066

TMTsixplex Label Reagent Set Labeling Reagents for Multiplexed and Absolute Protein Quantification

Kit

90067

TMTzero™ Label Reagent Labeling Reagent for Multiplexed and Absolute Protein Quantification

Kit

HeavyPeptides Kits are isotopically-labelled, nonradioactive custom peptides for mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy applications. They can be used as sequence-identical internal weight standards for quantitation of proteins and protein mixtures. Researchers can choose from a variety of custom-made HeavyPeptide kits to preform relative or absolute quantitation, including the AQUA technique3, developed by Harvard Medical School. Combined with our high performance range of MS instruments, such as the LTQ FT™, customers have access to the most powerful tools in Proteomics.

For more detailed kit information, please visit www.thermo.com/pierce

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

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Thermo Scientific Products for Protein Quantification by Mass Spectrometry Modified HeavyPeptides for Quantification of Phosphorylated Proteins

Tryptic digestion

HeavyPeptide Spiking

LC separation

HeavyPeptides can be prepared with covalent modifications (e.g. phosphorylation, methylation, acetylation, etc). These are chemically identical to the naturally occurring post-translational modifications. HeavyPeptides represent one of the most powerful tools for relative and absolute quantification of post-translational modified proteins by LC-MS. Applications: • Biomarker discovery and validation • Pharmacokinetics • Metabolomics • Clinical biochemistry (drug and metabolite monitoring) • Anti-doping testing • Protein expression • Validation of siRNA experiments • Cell signalling profiling For a more detailed explanation, please refer to www.thermo.com/ heavypeptide. To apply for your free demo kit please send an email to [email protected] using the code BROHP07. 1. BRIMS: Biomarker Research Initiatives in Mass Spectrometry 2. IP and Trademark: AQUA: This method was developed by Dr.Steve Gygi and colleagues at Harvard Medical School [Stemmann O, Zou H, Gerber SA, Gygi SP, Kirschner MW; Dual inhibition of sister chromatid separation at metaphase, Cell 2001, Dec 14, 107:715-726] and is the subject of both US and PCT Patent Applications. Limited noncommercial use of this method is permitted under a licensing arrangement with Harvard Medical School. AQUA is a trade mark of Harvard Medical School.

MS/MS analyses

Absolute quantification

References 1. Wienkoop, W. Weckwerth,: J. Exp. Botany , 2006;57(7): 1529-3 2. Aebersold, M. Mann, Nature 422, 198-207 (2003) 3. Arnott et al., Mol. Cell. Proteomics 1, 148-156 (2002) 4. Barnidge et al., Anal. Chem. 75, 445-51 (2003) 5. A. Gerber, J. Rush, 0. Stemman, M. W. Kirschner, S. P. Gygi, PNAS USA 100, 6940 ff. (2003) 6. Kuster et al, Nature Reviews, Molecular Cell Biology, Vol 6, 577-581 (2005) 7. Peng, S. P. Gygi, J. Mass Spectrom. 36, 1083 ff. (2001) 8. Stemmann, H. Zou, S.A. Gerber, S.P. Gygi, M.W. Kirschner, Cell 107, 715-726 (2001) 9. Washburn, D. Wolters, J. R. Yates, 3rd, Nat. Biotechnol. 19, 242 ff. (2001)

Native peptide HeavyPeptide AQUA

Figure 3. Thermo Scientific HeavyPeptide and LC-MS quantification. When a protein is enzymatically digested and analyzed by LC-MS some of the resulting peptides are unique identifiers of the protein. These are called signature peptides. When labeled with heavy isotopes (13C / 15N) and quantified by amino acid analysis (AAA) they are the ideal internal standard for protein quantification by mass spectrometry: HeavyPeptides AQUA. After spiking the sample containing the protein of interest with the HeavyPeptide it is analyzed by LCMS. The peaks of the light and the HeavyPeptide are detected and measured. From the comparison of the two peak areas it is possible to derive the quantity of the light peptide, and therefore the concentration of the protein in the sample.

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For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Instruments for Mass Spectrometry LTQ Orbitrap XL ETD Hybrid Mass Spectrometer The Thermo Scientific LTQ Orbitrap XL ETD™ mass spectrometer (LC-MS) is the most powerful instrument available for proteomics analyses, combining three different and complementary fragmentation techniques, CID, HCD and ETD, with the benefit of Orbitrap™ performance. This unique combination provides the most comprehensive solution available for complex PTM analysis, intelligent sequencing of peptides, top-down and middle-down analysis, and protein quantitation using label-free, metabolic, or stable isotope labelling techniques. The LTQ Orbitrap XL ETD hybrid mass spectrometer provides protein researchers: • Identification of unexpected PTMs using high resolution and accurate mass sample analysis • Absolute confidence for sequence assignments using multiple, complementary activation types • High-throughput sequencing applications with parallel MS acquisition capabilities • Quantitation of low abundance peptides with a wide dynamic MS range

Analysis of Post-translational Modifications (PTMs) Post-translational modifications to protein are the most important regulatory event in the operation of cells, and understanding the mechanism of these modifications requires the ability to identify the type, as well as the site of the PTM. The study of labile PTMs, such as glycosylation and phosphorylation, is limited by inadequate sequence information when using traditional fragmentation techniques, such as collisional induced dissociation (CID), because the modification is preferentially lost during peptide fragmentation. Electron transfer dissociation (ETD), conversely, is a soft fragmentation mechanism enabling highly-sensitive analyses of labile PTMs. Unparalleled Protein Sequence Coverage The automated Thermo Scientific Data Dependent™ Decision Tree selects the optimal fragmentation technique (based on the peptide’s charge state, molecular weight, m/z, etc.) to achieve the optimal fragmentation efficiency. Complementary ETD, CID and HCD fragmentation produces a higher sequence coverage for unambiguous protein characterization and de novo sequencing. Parallel acquisition (in both the linear ion trap and Orbitrap mass analyzers) permits the identificaion of the largest number of peptides and proteins possible within a single analytical run. Top-down, Middle-down Protein Analysis The Thermo Scientific top-down solution enables the definitive identification and characterization of intact proteins and their post-translational modifications. The resolution and accuracy of the LTQ Orbitrap XL ETD Mass Spectometer enables the unambiguous charge state determination of precursor and fragmentation ions. This data is used by ProSightPC™ and ProMass Deconvolution™ software suites for the analysis of intact proteins and large peptides and oligonucleotides.

For more information on the LTQ Orbitrap XL ETD mass spectrometer, visit www.thermo.com/orbitrap or email [email protected]

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

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Thermo Scientific Instruments for Mass Spectrometry MALDI LTQ XL Mass Spectrometer The Thermo Scientific MALDI LTQ XL™ Mass Spectrometer provides scientists with an MSn solution for analyzing whole tissue, biological and polymer samples without extensive sample preparation. A sensitive linear ion trap with MALDI (matrix-assisted laser desorption/ionization) capabilities provides faster sample preparation and analysis, providing data complementary to electrospray ionization (ESI) for more reliable and definitive answers for protein identification and structural elucidation. The addition of MALDI to the LTQ XL linear ion trap provides information-rich spectra that are essential for the analysis of digested proteins, peptides and post-translational modifications. The MALDI LTQ XL mass spectrometer solution provides: • High throughput analysis of in-gel digests for protein ID • Direct tissue analysis by MALDI MS imaging • Thermo Scientific ImageQuest™ Software for visualization of tissue imaging data Tissue Imaging The Thermo Scientific tissue-imaging solution provides researchers with the unparalleled sensitivity and unmatched MSn spectral quality of linear ion trap technology, as well as faster sample analysis compared to the standard practice of homogenizing and extracting the biomolecules from tissue. ImageQuest software is used for visualization of imaging data, and for creating two-dimensional and three-dimensional maps of analyzed tissue, offering a complete package for the acquisition and presentation of MS images directly from tissue samples.

TSQ Quantum Ultra Triple Quadrupole Mass Spectrometer The Thermo Scientific TSQ Quantum Ultra™ triple quadrupole mass spectrometer provides simultaneous qualitative and quantitative targeted protein analysis. Using the highly selective reaction monitoring (H-SRM) assay for peptide detection and confirmation enables much lower detection limits than the traditional approach for peptide detection and identification using SRM-triggered MS/MS data acquisition. This provides a wider quantitative dynamic range, essential for trace-level biomarkers. The TSQ Quantum Ultra mass spectrometer delivers: • Excellent sensitivity, analytical assay precision and quantitative accuracy for targeted protein quantitation • Wide dynamic range and excellent quantitative accuracy for targeted peptide quantitation • Higher resolution precursor ion isolation and faster cycle time for the identification of the myriad targeted peptides • Elimination of non-specific interference from sample matrix Targeted Protein Quantitation The intuitive user interface of the SRM Workflow allows a rapid progression from biomarker discovery to targeted protein quantitation experiments. Pre-defined and user-defined parameters dramatically increase the ability to create a proteotypic peptide list, building a clinically useful, high throughput, and highly selective assays.

For more information on the TSQ Quantum Ultra mass spectrometer, visit www.thermo.com/tsq or email [email protected]

For more information on the MALDI LTQ XL mass spectrometer, visit www.thermo.com/maldi or email [email protected]

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For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Technical Handbooks & Guides

Cell Lysis Technical Handbook

RED Device Brochure

This 45-page handbook provides protocols, technical tips and product information to help maximize results for Protein/Gene Expression studies. The handbook provides background, Thermo Scientific Pierce helpful hints and troubleshooting Cell Lysis Technical Handbook advice for cell lysis, protein purification, cell fractionation, protease inhibitors and protein refolding. The handbook is an essential resource for any laboratory studying Protein/Gene Expression. To request a free copy, log on to www.thermo.com/pierce or call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

Rapid Equilibrium Dialysis (RED) Device is a transforming technology for plasma protein binding assays. Learn more about this technology and how it can accelerate lead optimization and reduce attrition rate in this brochure. Also learn about applications in drug partition studies and protein binding in liver micorsomes. To request a free copy, log on to www.thermo.com/pierce or call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

Dialysis and Desalting Technical Handbook

Protein Purification Technical Handbook

This updated 23-page handbook features the popular Thermo Scientific Slide-A-Lyzer Dialysis Cassettes, SnakeSkin Dialysis Tubing and Zeba Protein Desalt Products. The handbook presents numerous tips to improve usage of these products, as well as helpful selection criteria to choose the most appropriate tool for your application. To request a free copy, log on to www.thermo.com/pierce or call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

This updated 79-page handbook provides technical and product information to help maximize results for affinity purification procedures. The handbook provides background, helpful hints and troubleshooting advice for covalent coupling of affinity ligands to chromatography supports, avidin:biotin-binding, affinity purification of antibodies, immunoprecipitation and coimmunoprecipitation assays, affinity procedures for contaminant removal, and related procedures. To request a free copy, log on to www.thermo.com/pierce or call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

Tandem Mass Tags and methods of their use are protected by pending patent applications and granted patents worldwide including European Patent EP # 1,275,004, United States Patent # 7,294,456 and United States Patent Application 10/489,341. Thermo Scientific SuperSignal Technology is protected by U.S. Patent # 6,432,662. Thermo Scientific SwellGel Technology is protected by U.S. Patent # 6,709,743. Thermo Scientific B-PER Technology is protected by U.S. Patent # 6,174,704. U.S. patent pending on Mitochondria Isolation Kit Technology. U.S. patent pending on Zeba Micro Column Technology and Imperial Protein Stain Technology.

Tandem Mass Tag, TMT, TMTzero™, TMTsixplex™ and TMTduplex™ are trademarks of Proteome Sciences plc. Triton® is a registered trademark of Rohm & Haas Company. Cibacron™ is a trademark of Ciba Specialty Chemicals. SimplyBlue™ is a trademark of Invitrogen Life Sciences. EZBlue™ is a trademark of Sigma-Aldrich. Bio-Safe™ is a trademark of Bio-Rad Laboratories. Tween® is a trademark of ICI Americas. Voyager-DE™ is a trademark of Applied Biosystems. LabChip® is a trademark of Caliper Technologies. Optiprep® is a trademark of Axis-Shield P C AS. AQUA® is a trademark of Harvard Medical School. Agilent® is a trademark of Agilent Technologies, Inc. ProSightPC™ is a trademark of the University of Illinois at Urbana-Champaign. ProMass™ is a trademark of Novatia, LLC.

®

Fea ur ng Ce l Lys s Reagen s and Deterg n s

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

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Contact Information Belgium and Europe, the Middle East and Africa Distributors Tel: +32 53 85 71 84 France Tel: 0 800 50 82 15 The Netherlands Tel: 076 50 31 880 Germany Tel: 0228 9125650 United Kingdom Tel: 0800 252 185 Switzerland Tel: 0800 56 31 40 Email: [email protected] www.thermo.com/perbio United States

1601518 07/08

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