Fluorescent Product Guide
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Th no Pie er w rc m so e ® o ld Sc a ie s nt ifi c
Thermo Scientific Pierce Fluorescent Products Guide
Featuring • Thermo Scientific DyLight™ Dyes and Conjugates, Dye Removal Columns, Antibody Labeling Kits, Western Blotting Kits, and MW Markers
Thermo Scientific Fluorescent Products
Table of Contents DyLight Dye Summary.............................................................................................. 3 DyLight Amine- and Sulfhydryl-Reactive Dyes ........................................................ 4 NHS Ester and Maleimide DyLight Dye Removal Columns................................................................................ 7 Antibody Labeling Kits............................................................................................ 8 Microscale and Regular Antibody Labeling Kits DyLight Conjugates................................................................................................. 10 DyLight Fluorescent Protein Molecular Weight Markers .......................................... 12 Infrared and Fluorescent Protein Molecular Weight Markers DyLight Western Blotting Kits ................................................................................. 14 Krypton Fluorescent Protein Stain........................................................................... 16
2
Thermo Scientific DyLight Dye Summary
Bright New Alternatives to Alexa®, CyDye® and LI-COR Fluorescent Dyes Thermo Scientific DyLight Dyes have absorption spectra ranging from 400 nm to 770 nm (Table 1) and match the principal output wavelengths of common fluorescence instrumentation. The DyLight Dyes exhibit higher fluorescence intensity and photostability than Alexa, CyDye and LI-COR Dyes in many applications and remain highly fluorescent over a broad pH range (pH 4-9). Additionally, the water solubility of the DyLight Dyes allows a high dye-to-protein ratio without precipitation during conjugation. Table 1. Spectral properties of Thermo Scientific DyLight Fluorescent Dyes. Emission Blue Green Yellow Red Red Near Infrared
DyLight Dye 405 488 549 633 649 680 800
Ex/Em* 400/420 493/518 550/568 638/658 646/674 682/715 770/794
ε† 30,000 70,000 150,000 170,000 250,000 140,000 270,000
Spectrally Similar Dyes Alexa 405 and Cascade Blue Alexa 488, fluorescein and FITC Alexa 546, Alexa 555, Cy3 and TRITC Alexa 633 Alexa 647 and Cy5 Alexa 680 and Cy5.5 IRDye 800
*Excitation and emission maxima in nanometers †Molar extinction coefficient (M-1 cm-1)
Figure 1. Thermo Scientific DyLight 488 and DyLight 633 Dyes exhibit outstanding fluorescence in structured illumination. The uniform fluorescence intensity throughout the images demonstrates the outstanding brightness and photostability of DyLight 488 and 633 Dyes. Red: Alpha tubulin detected in HeLa cells with anti-tubulin monoclonal antibody and DyLight 633 Dye-conjugated secondary antibody (highly cross-adsorbed). Green: Histone H4 detected with anti-histone monoclonal antibody and DyLight 488 Dye-conjugated secondary antibody (highly cross-adsorbed). Blue: Nucleus counter-stained with fluorescent mounting media containing DAPI. Images were acquired with the Axio Imager.Z1 and ApoTome Slider (Zeiss MicroImaging, Inc). The ApoTome Module provides confocal-like resolution allowing optical sectioning without using a pinhole (e.g., confocal). No image enhancement was performed.
3
Dyes
Thermo Scientific DyLight Amine-Reactive and Sulfhydryl-Reactive Dyes Excellent photostability make these dyes the clear alternative
Highlights: • Available in both amine- and sulfhydryl-reactive chemistries for fast and efficient labeling of IgG or other proteins • High water solubility • Excellent photostability • Compatible with common fluorescence instrumentation
Applications: • Fluorescence microscopy • Western blot detection • Protein arrays • Flow cytometry • ELISA • FRET-based technology • And many more
4
DyLight 405
400
450
500
550
550
DyLight 680
520
570
620
670
550
600
700
750
800
850
900
DyLight 800
600
650
Excitation
Emission
Excitation
Abs max: 550 nm Em max: 568 nm
550
650
Wavelength (nm)
DyLight 549
500
800
Abs max: 682 nm Em max: 715 nm
Wavelength (nm)
450
750
Emission
Emission 470
700
DyLight 488
Excitation
420
650
Wavelength (nm)
Abs max: 493 nm Em max: 518 nm
370
600
Wavelength (nm)
Abs max: 770 nm Em max: 794 nm
650
700
700
Emission
350
Excitation
300
Abs max: 646 nm Em max: 674 nm
Excitation
Emission
Excitation
Abs max: 400 nm Em max: 420 nm
Emission
DyLight 649
750
800
Wavelength (nm)
Wavelength (nm)
DyLight 633
Emission
Excitation
Abs max: 638 nm Em max: 658 nm
500
550
600
650
700
750
Wavelength (nm)
Figure 1. Excitation and emission spectra for Thermo Scientific DyLight Dyes.
5
Dyes (Cont.)
Ordering Information Product # Description Amine-Reactive Dyes 46400 DyLight 405 NHS Ester 46401 DyLight 405 NHS Ester 46402 DyLight 488 NHS Ester 46403 DyLight 488 NHS Ester 46407 DyLight 549 NHS Ester 46408 DyLight 549 NHS Ester 46414 DyLight 633 NHS Ester 46417 DyLight 633 NHS Ester 46415 DyLight 649 NHS Ester 46416 DyLight 649 NHS Ester 46418 DyLight 680 NHS Ester 46419 DyLight 680 NHS Ester 46421 DyLight 800 NHS Ester 46422 DyLight 800 NHS Ester Sulfhydryl-Reactive Dyes 46660 DyLight 405 Maleimide 46602 DyLight 488 Maleimide 46607 DyLight 549 Maleimide 46613 DyLight 633 Maleimide 46615 DyLight 649 Maleimide 46618 DyLight 680 Maleimide 46621 DyLight 800 Maleimide
6
Pkg. Size
U.S. Price
1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg
$199 $159 $199 $159 $199 $159 $199 $159 $199 $159 $199 $159 $199 $159
1 mg 1 mg 1 mg 1 mg 1 mg 1 mg 1 mg
$199 $199 $199 $199 $199 $199 $199
Dye Removal Columns
High-performance dye removal columns Convenient new options for fluorescent antibody labeling The same high performance dye removal spin-columns used in the Thermo Scientific DyLight Antibody Labeling Kits are now available separately.
Highlights: • Fast – remove free dye from conjugates in approximately 30 seconds • Efficient – purification resin provides outstanding conjugate recovery (Figure 1) • Minimal sample dilution
100%
Conjugate Recovery
80%
60%
40%
85%
84%
84%
(n = 12)
(n = 12)
(n = 12)
FITC 79% 90% 85%
Fluorescein 86% 81% 84%
Rhodamine 94% 74% 84%
20%
0%
Conjugate Goat anti-mouse IgG Streptavidin Average Recovery
Figure 1. Thermo Scientific DyLight Dye Removal Columns provide outstanding conjugate recovery.
Ordering Information Product # 22858
Description Dye Removal Columns
Pkg. Size Kit
Includes: Purification Resin Spin Columns Microcentrifuge Collection Tubes
5 ml 10 ea. 20 ea.
U.S. Price $ 99
7
Antibody Labeling Kits
Label and purify antibodies in one hour Antibody labeling kits for fast and efficient labeling and exceptional recovery The Thermo Scientific DyLight Antibody Labeling Kits were specifically developed for fast, efficient labeling of antibodies. Two convenient kit formats are available to accommodate varied labeling requirements. The Antibody Labeling Kits contain all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The DyLight Microscale Antibody Labeling Kits contain all the necessary components to perform five separate labeling reactions using 100 µg of IgG. The labeling kits use high-performance spin desalting columns to provide exceptional dye removal and antibody recovery (Figure 1).
Highlights: • Fast – fluorescently label and purify protein in approximately one hour • Amine-reactive dyes – label virtually any protein • Pre-measured fluorescent dye – eliminate the time, waste and hassle associated with weighing dye
• Efficient non-reacted dye removal • Minimal sample dilution • Spin column format eliminates the need for column preparation, fraction screening and waiting for protein to emerge from column • Easy protocol (Figure 2)
Antibody Recovery
100
Figure 1. Thermo Scientific DyLight Antibody Labeling Kits provide outstanding recovery. The percent recovery for DyLight Antibody Labeling Kits is the average for 16 labeling reactions using three different antibodies. The percent recovery for DyLight Microscale Antibody Labeling Kits is the average for 13 labeling reactions using three different antibodies.
80 60 40
81%
75%
Thermo Scientific DyLight Antibody Labeling Kit
Thermo Scientific DyLight Microscale Antibody Labeling Kit
20 0
Table 1. Spectral properties of Thermo Scientific DyLight Fluorescent Dyes. Emission Blue Green Yellow Red Red Near Infrared
DyLight Dye 405 488 549 633 649 680 800
Ex/Em* 400/420 493/518 550/568 638/658 646/674 682/715 770/794
*Excitation and emission maxima in nanometers †Molar extinction coefficient (M-1 cm-1)
8
ε† 30,000 70,000 150,000 170,000 250,000 140,000 270,000
Spectrally Similar Dyes Alexa 405 and Cascade Blue Alexa 488, fluorescein and FITC Alexa 546, Alexa 555, Cy3 and TRITC Alexa 633 Alexa 647 and Cy5 Alexa 680 and Cy5.5 IRDye 800
Step 1. Labeling reaction
Step 2. Removal of excess fluorescent dye
Centrifuge 30 seconds
Add antibody to vial containing pre-measured dye. Incubate 1 hour at room temperature.
Apply labeling reaction to Spin Desalting Column.
Recover labeled antibody.
Figure 2. Protocol summary for Thermo Scientific DyLight Antibody Labeling Kits.
Microscale Kits Contain sufficient reagents to label and purify 5 x 100 µg of IgG In addition to contents listed below, all Microscale Kits include: • Reaction Buffer, 1 ml • Microcentrifuge Collection Tubes, 10 each • Spin Columns, 5 each • Purification Resin, 5 ml
Ordering Information Product # 53021
Description DyLight 405 Microscale Antibody Labeling Kit
Pkg. Size Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials
DyLight 405 NHS Ester
53025
DyLight 488 Microscale Antibody Labeling Kit DyLight 488 NHS Ester
53035
DyLight 549 Microscale Antibody Labeling Kit DyLight 549 NHS Ester
53047
DyLight 633 Microscale Antibody Labeling Kit DyLight 633 NHS Ester
53051
DyLight 649 Microscale Antibody Labeling Kit DyLight 649 NHS Ester
53057
DyLight 680 Microscale Antibody Labeling Kit DyLight 680 NHS Ester
53063
DyLight 800 Microscale Antibody Labeling Kit DyLight 800 NHS Ester
U.S. Price $275 $275 $275 $275 $275 $275 $275
Antibody Labeling Kits Contain sufficient reagents to label and purify 3 x 1 mg of IgG or similar quantities of other proteins In addition to contents listed below, all Antibody Labeling Kits include: • Reaction Buffer, 1 ml • Spin Columns, 6 each
• Microcentrifuge Collection Tubes, 12 each • Purification Resin, 5 ml
Ordering Information Product # 53020
Description DyLight 405 Antibody Labeling Kit DyLight 405 NHS Ester
53024
DyLight 488 Antibody Labeling Kit DyLight 488 NHS Ester
53034
DyLight 549 Antibody Labeling Kit DyLight 549 NHS Ester
53046
DyLight 633 Microscale Antibody Labeling Kit DyLight 633 NHS Ester
53050
DyLight 649 Antibody Labeling Kit DyLight 649 NHS Ester
53056
DyLight 680 Antibody Labeling Kit DyLight 680 NHS Ester
53062
DyLight 800 Antibody Labeling Kit DyLight 800 NHS Ester
Pkg. Size Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials
U.S. Price $275 $275 $275 $275 $275 $275 $275
9
Conjugates
Thermo Scientific DyLight Conjugates Excellent brightness make these conjugates a clear alternative
Highlights: • Available conjugated to commonly used secondary antibodies, streptavidin and NeutrAvidin Biotin-Binding Protein • Molar ratio (dye:protein) optimized to provide excellent fluorescent intensity • Stable for 1 year at 4°C • Antibody conjugates are affinity-purified to minimize cross-reactivity
Western Blotting Figure 1. Two-color infrared Western blot detection of p53 and cyclophilin B knockdown using Thermo Scientific DyLight 680- and Thermo Scientific DyLight 800-labeled secondary antibodies. Protein lysate from transfected A549 cells was separated using SDS-PAGE and transferred to PVDF membrane. Lane 1: MW marker, Lane 2: mock transfected sample, Lane 3: negative control siRNA, Lane 4: siRNA targeted against p53 and Lane 5: siRNA targeted against cyclophilin. The membranes were imaged with the Odyssey Infrared Imaging System using the 700 and 800 channels.
1
10
2 3
4
5
Microplate-Based Assays
Relative Fluorescent Intensity
60,000
Figure 2. Thermo Scientific DyLight 649 Streptavidin and Thermo Scientific NeutrAvidin Conjugates are brighter than Alexa 647 Streptavidin Conjugates in microplate-based assays. Microplates were coated with biotinylated BSA at the indicated concentrations. DyLight 649 and Alexa 647 Conjugates were diluted 1:250 (from 1 mg/ml stock solutions) in PBS and 100 µl was applied to each plate. Fluorescent intensity was measured with a Tecan Safire™ Microplate Reader using Cy5 Dye settings.
50,000 40,000 30,000
20,000 DyLight 649 Streptavidin DyLight 649 NeutrAvidin Alexa 647 Streptavidin
10,000 0 0
50
100
150
200
250
Biotinylated BSA (ng/well)
Relative Fluorescent Intensity
50,000
Figure 3. Thermo Scientific DyLight 549 Goat AntiMouse Conjugates are brighter than Alexa 555 Dye conjugates in microplate-based assays. Microplates were coated with Mouse IgG at the indicated concentrations. DyLight 549 and Alexa 555 Conjugates were diluted 1:250 (from 1 mg/ml stock solutions) in PBS and 100 µl was applied to each plate. Fluorescent intensity was measured with a Tecan Safire Microplate Reader using Cy3 Dye settings.
45,000 40,000 35,000 30,000 25,000 20,000 15,000 DyLight 549 Goat anti-mouse IgG
10,000
Alexa 555 Goat anti-mouse IgG
5,000 0
0
200
400
600
800
1,000
1,200
Mouse IgG (ng/well)
Relative Fluorescent Intensity
16,000,000
Figure 4. Thermo Scientific DyLight 680 and Thermo Scientific DyLight 800 Streptavidin Conjugates are brighter than Alexa 680 or IRDye 800 Conjugates in microplate-based assays. Microplates were coated with biotinylated BSA at the indicated concentrations. Conjugates were diluted to 10 µg/ml in PBS and 100 µl was applied to each plate. Fluorescent intensity was measured with the Odyssey Infrared Imaging System using the 700 and 800 channels.
DyLight 800 Streptavidin IRDye 800 Streptavidin 12,000,000 DyLight 680 Streptavidin Alexa 680 Streptavidin
8,000,000
4,000,000
0
2
4 6 8 Biotinylated BSA (ng per well)
10
12
Ordering Information Conjugates: Package size for these items is 1 mg at 1 mg/ml. Product # / U.S. Price DyLight DyLight DyLight Description 488 Dye 549 Dye 649 Dye Goat Anti-Mouse 35502/$128 35507/$128 35515/$128 IgG (H+L) Goat Anti-Rabbit 35552/$128 35557/$128 35565/$128 IgG (H+L) Streptavidin 21832/$155 21837/$155 21845/$155 NeutrAvidin 22832/$170 22837/$170 22845/$170 Biotin-Binding Protein
DyLight DyLight 680 Dye 800 Dye 35518/$128 35521/$128 35568/$128 35571/$128 21848/$155 21851/$155 22848/$170 22853/$170
11
Fluorescent Protein Molecular Weight Markers
Fluorescent MW markers for Western blotting and in-gel detection One- or two-color fluorescence detection with one protein molecular weight marker Fluorescent Thermo Scientific DyLight Infrared Protein Molecular Weight Markers have been optimized for direct visualization of marker proteins after SDS-polyacrylamide gel electrophoresis (PAGE) (Figure 1). Two convenient formats are available to accommodate fluorescence imaging requirements in the visible or near-infrared region of the spectrum. Each protein in the mixture is labeled with two fluorescent dyes to provide flexible one- or two-color detection with common fluorescence imaging systems (Table 1). The markers are compatible with Western blotting (Figure 2) and can be detected by virtually any in-gel staining method (Figure 3). The DyLight Protein Molecular Weight Markers consist of nine proteins with molecular weights in the range of 6K to 200K.
Highlights: • Easily multiplexed – two excitation and emission maxima enable one- or two-color fluorescence detection • Saves time – eliminate the need for awkward marking or overlay procedures • Fluorescence and colorimetric – two detection options available: in-gel or on-membrane
• Instrument-compatible – spectra are compatible with common fluorescence imaging systems • Photostable – capture multiple images with no decrease in fluorescent intensity
Table 1. Spectral characteristics of Thermo Scientific DyLight Fluorescent Protein Molecular Weight Markers.
DyLight 547 Fluors DyLight 647 Fluors
12
Excitation (nm) 557 652
Emission (nm) 574 673
Extinction Coefficient (min) 150,000 M-1 cm-1 250,000 M-1 cm-1
Figure 1 A B
Figure 2 A B
Figure 3 A B
Myosin (200K) Phosphoylase B (97K) BSA (66K) Protein A (42K) Protein L (36K) Peanut Agglutinin (28K) Trypsin Inhibitor (20K) Lysozyme (14K) Aprotinin (6K)
Fluorescent and Colorimetric Detection of Thermo Scientific DyLight Fluorescent Protein Molecular Weight Markers Figure 1. Panel A. Direct in-gel fluorescent detection. Marker proteins (5 µl) were separated in 4-20% Tris-glycine gels. The gels were imaged with the Kodak Image Station 2000MM using a five-minute exposure at f2.8 with the A. 535/600 nm excitation/emission filter set and B. 625/700 nm excitation/emission filter set. Figure 2. Fluorescent Western blot detection. Marker proteins (5 µl) were separated in 4-20% Tris-glycine gels and transferred to A. nitrocellulose or B. PVDF membrane. Blots were imaged with the Typhoon 9410 at 500V PMT using the A. Cy3 Fluor and B. Cy5 Fluor laser settings. Note: Proteins in the marker mix produce uniform fluorescent intensities in SDS-PAGE applications; however, variations in protein-transfer efficiency affect fluorescent intensity. For example, high molecular weight proteins, such as myosin (200K), typically transfer less efficiently than low molecular weight proteins. Figure 3. Colorimetric in-gel detection. Marker proteins (10 µl) were separated in 4-20% Tris-glycine gels and stained with A. Thermo Scientific Imperial Protein Stain† and B. silver stain.
Ordering Information Product # 22859
Description DyLight Infrared Protein Molecular Weight Markers
Pkg. Size 250 µl
U.S. Price $199
250 µl
$199
Sufficient material for loading 50 gel lanes
26665
DyLight Fluorescent Protein Molecular Weight Markers
Sufficient material for loading 50 gel lanes. † See patent information on inside back cover
13
Western Blotting Kits
Multiplex Fluorescent Western Blotting Kits Fluorescent Western blotting made easy The fluorescence detection of two different targets on a single Western blot is now easier to perform with the new Thermo Scientific DyLight 680/800 and 549/649 Western Blotting Kits. The kit provides a highly optimized and convenient format to save you the time and frustration of having to evaluate reagents for compatibility with fluorescent Western blotting (Figure 1). Each kit contains sufficient reagents for 10 Western blots and includes the new Thermo Scientific Pierce Dual-Labeled Protein Molecular Weight Markers† and secondary antibodies conjugated to DyLight 680 and 800 Fluorescent Dyes (Table 1). The DyLight 680/800 Western Blotting Kit uses DyLight 680 and DyLight 800 labeled secondary antibodies for imaging in the near-infrared region (e.g. LI-COR Odyssey Imaging System) of the spectrum (Figure 2). Highlights: • Easily multiplexed – detect two different targets on a single Western blot • Optimized format – protocol requires little to no optimization, saving time and the frustration of having to evaluate and optimize reagents (e.g., secondary antibody dilutions, etc.) • Plug-and-play – kits include DyLight-labeled secondary antibodies, blocking buffer, wash buffer, dual-labeled fluorescent molecular weight markers and low-fluorescence membranes
• Instrument-compatible – DyLight Fluor spectra match the principal output wavelengths of common fluorescence instrumentation • Optimized format – provides low background and high signal intensity • Convenient – saves time and money associated with optimizing fluorescent Western blots
Table 1. Spectral properties of Thermo Scientific DyLight Western Blotting Kits.
12 ng
6 ng
3 ng
1 ng
0.5 ng
12 ng 6 ng 3 ng 1 ng
25 ng
*Excitation and emission maxima in nanometers †Molar extinction coefficient (M-1 cm-1)
Ex/Em*
ε†
Spectrally Similar Dyes
550/568 646/674
150,000 250,000
Alexa 546, Alexa 555, Cy3 and TRITC Alexa 647 and Cy5
682/715 770/794
140,000 270,000
Alexa 680 and Cy5.5 IRDye 800
25 ng MW Marker
Western Blotting Kit DyLight 549/649 Western Blotting Kit DyLight 549 Goat Anti-Mouse IgG (H+L) DyLight 649 Goat Anti-Rabbit IgG (H+L) DyLight 680/800 Western Blotting Kit DyLight 680 Goat Anti-Mouse IgG (H+L) DyLight 800 Goat Anti-Rabbit IgG (H+L)
Tubulin
25 ng
12 ng
6 ng
3 ng
1 ng
0.5 ng
12 ng 6 ng 3 ng 1 ng
25 ng
ECL Plex Western Blotting Kit
Tubulin
TNFα
14
DyLight 549/649 Western Blotting Kit
MW Marker
TNFα
Figure 1. The Thermo Scientific DyLight 549/649 Western Blotting Kit provides lower background and higher signal in two-color Western blot detection compared to a competing fluorescent Western blotting kit. Proteins were separated in 4-20% Precise Protein Gels and transferred to low-fluorescence PVDF membrane. The membranes were blocked overnight in 1% BSA and target proteins were detected following manufacturer-recommended protocols. Membranes were imaged with the Typhoon 9410.
50 µg
25 µg
12.5 µg
6.25 µg
0.063 µg
0.125 µg
0.25 µg
0.5 µg
1 µg
Figure 2. The Thermo Scientific DyLight 680/800 Western Blotting Kit provides low background and high signal in two-color Western blot detection. Proteins were separated in 4-20% Thermo Scientific Precise Protein Gels and transferred to LowFluorescence PVDF Transfer Membrane (Product # 22860). The membrane was blocked overnight in SEA BLOCK Blocking Buffer (Product # 37527) and target proteins were detected following the recommended protocol. Membranes were imaged with the LI-COR Odyssey Infrared Imaging System. Tubulin was detected from the indicated quantity of HeLa cell lysate. Purified TNFα was detected at the indicated quantity.
Tubulin
TNFα
Ordering Information Product # 22855
22854
Description DyLight 680/800 Western Blotting Kit
Pkg. Size Kit
Sufficient reagents for 10 Western blots. Includes: DyLight 680 Goat Anti-Mouse IgG (H+L) DyLight 800 Goat Anti-Rabbit IgG (H+L) DyLight Infrared Protein Molecular Weight Markers Wash Buffer (30X) SEA BLOCK Blocking Buffer Low-Fluorescence PVDF Transfer Membrane
120 µl 120 µl 30 µl 200 ml 500 ml 10 ea.
DyLight 549/649 Western Blotting Kit
Kit
Sufficient reagents for 10 Western blots. Includes: DyLight 549 Goat Anti-Mouse IgG (H+L) DyLight 649 Goat Anti-Rabbit IgG (H+L) Fluorescent Dual-labeled Protein MW Markers Wash Buffer (30X) Blocker BSA in PBS (10X) Low-Fluorescence PVDF Transfer Membrane
50 µl 50 µl 60 µl 200 ml 50 ml 10 ea.
Complementary Products Product # Description 22859 DyLight Infrared Protein Molecular Weight Markers
U.S. Price $199
$199
Pkg. Size 250 µl
U.S. Price $199
10/pkg.
$ 78
250 µl 500 ml Kit
$199 $129 $275
Kit
$275
Kit
$275
Kit
$275
Sufficient material for loading 50 gel lanes.
22860 26665 37527 53056
Low-Fluorescence PVDF Transfer Membrane, 0.2 µm 7 cm x 8.4 cm DyLight Dual Fluor-Labeled Protein MW Markers SEA BLOCK Blocking Buffer DyLight 680 Antibody Labeling Kit Sufficient reagents to label and purify 3 x 1 mg of IgG
53057
DyLight 680 Microscale Antibody Labeling Sufficient reagents to label and purify 5 x 100 µg of IgG.
53062
DyLight 800 Antibody Labeling Kit Sufficient reagents to label and purify 3 x 1 mg of IgG.
53063
DyLight 800 Microscale Antibody Labeling Kit
Sufficient reagents to label and purify 5 x 100 µg of IgG. † See patent information on inside back cover
15
Fluorescent Dye-Based Stains
Thermo Scientific Krypton Fluorescent Protein Stain A faster, affordable fluorescent stain that provides excellent performance Thermo Scientific Krypton Protein Stain† is a fluorescent stain for detecting proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D gels.
Highlights • Excitation/emission maxima – 520/580 nm • Compatibility – works with all SDS-polyacrylamide and 2-D gel types and with MS analysis • Linear quantitative range – three to four orders of magnitude • Sensitive – detects down to 0.25 ng protein with the basic 2.7-hour protocol • Fast – using the rapid protocol, detects down to 2 ng protein in 30 minutes • Comparative – minimal differential staining of proteins
Ordering Information Product # 46628
Description Krypton Protein Stain (10X)
Pkg. Size 20 ml
U.S. Price $ 39
100 ml
$110
500 ml
$399
Sufficient reagent to stain four mini gels (8 cm x 10 cm)
46629
Krypton Protein Stain (10X) Sufficient reagent to stain 20 mini gels (8 cm x 10 cm) or two to four large-format gels
46630
Krypton Protein Stain (10X)
Sufficient reagent to stain 100 mini gels (8 cm x 10 cm) or 10 to 20 large-format gels † See patent information on inside back cover
16
www.thermo.com/dylight
Pierce Protein Research Products Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316 • Customer Assistance E-mail: [email protected] Outside the United States, visit our website or call 815-968-0747 to locate your local branch office or distributor. © 2007 Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Prices listed herein were accurate at the time of printing. Visit our website for up-to-date prices. † U.S. patent pending on Dual-labeled Fluorescent Molecular Weight Marker Technology. U.S. patent pending on Imperial Protein Stain Technology. U.S. patent pending on Krypton Infrared Protein Stain Technology. Alexa® is a trademark of Molecular Probes, Inc. CyDye®, Cy® and Typhoon® are trademarks of GE Healthcare. Odyssey® is a trademark of LI-COR Biosciences. All other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries.
19
1601552 11/07
61105-0117
PO Box 117
• High water solubility • Excellent photostability
Thermo Scientific DyLight Fluors
Rockford, IL
Pierce Protein Research Products
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PRSRTD STD U.S. POSTAGE
Thermo Scientific Pierce Fluorescent Products Guide
Featuring • Thermo Scientific DyLight™ Dyes and Conjugates, Dye Removal Columns, Antibody Labeling Kits, Western Blotting Kits, and MW Markers
Thermo Scientific Fluorescent Products
Table of Contents DyLight Dye Summary.............................................................................................. 3 DyLight Amine- and Sulfhydryl-Reactive Dyes ........................................................ 4 NHS Ester and Maleimide DyLight Dye Removal Columns................................................................................ 7 Antibody Labeling Kits............................................................................................ 8 Microscale and Regular Antibody Labeling Kits DyLight Conjugates................................................................................................. 10 DyLight Fluorescent Protein Molecular Weight Markers .......................................... 12 Infrared and Fluorescent Protein Molecular Weight Markers DyLight Western Blotting Kits ................................................................................. 14 Krypton Fluorescent Protein Stain........................................................................... 16
2
Thermo Scientific DyLight Dye Summary
Bright New Alternatives to Alexa®, CyDye® and LI-COR Fluorescent Dyes Thermo Scientific DyLight Dyes have absorption spectra ranging from 400 nm to 770 nm (Table 1) and match the principal output wavelengths of common fluorescence instrumentation. The DyLight Dyes exhibit higher fluorescence intensity and photostability than Alexa, CyDye and LI-COR Dyes in many applications and remain highly fluorescent over a broad pH range (pH 4-9). Additionally, the water solubility of the DyLight Dyes allows a high dye-to-protein ratio without precipitation during conjugation. Table 1. Spectral properties of Thermo Scientific DyLight Fluorescent Dyes. Emission Blue Green Yellow Red Red Near Infrared
DyLight Dye 405 488 549 633 649 680 800
Ex/Em* 400/420 493/518 550/568 638/658 646/674 682/715 770/794
ε† 30,000 70,000 150,000 170,000 250,000 140,000 270,000
Spectrally Similar Dyes Alexa 405 and Cascade Blue Alexa 488, fluorescein and FITC Alexa 546, Alexa 555, Cy3 and TRITC Alexa 633 Alexa 647 and Cy5 Alexa 680 and Cy5.5 IRDye 800
*Excitation and emission maxima in nanometers †Molar extinction coefficient (M-1 cm-1)
Figure 1. Thermo Scientific DyLight 488 and DyLight 633 Dyes exhibit outstanding fluorescence in structured illumination. The uniform fluorescence intensity throughout the images demonstrates the outstanding brightness and photostability of DyLight 488 and 633 Dyes. Red: Alpha tubulin detected in HeLa cells with anti-tubulin monoclonal antibody and DyLight 633 Dye-conjugated secondary antibody (highly cross-adsorbed). Green: Histone H4 detected with anti-histone monoclonal antibody and DyLight 488 Dye-conjugated secondary antibody (highly cross-adsorbed). Blue: Nucleus counter-stained with fluorescent mounting media containing DAPI. Images were acquired with the Axio Imager.Z1 and ApoTome Slider (Zeiss MicroImaging, Inc). The ApoTome Module provides confocal-like resolution allowing optical sectioning without using a pinhole (e.g., confocal). No image enhancement was performed.
3
Dyes
Thermo Scientific DyLight Amine-Reactive and Sulfhydryl-Reactive Dyes Excellent photostability make these dyes the clear alternative
Highlights: • Available in both amine- and sulfhydryl-reactive chemistries for fast and efficient labeling of IgG or other proteins • High water solubility • Excellent photostability • Compatible with common fluorescence instrumentation
Applications: • Fluorescence microscopy • Western blot detection • Protein arrays • Flow cytometry • ELISA • FRET-based technology • And many more
4
DyLight 405
400
450
500
550
550
DyLight 680
520
570
620
670
550
600
700
750
800
850
900
DyLight 800
600
650
Excitation
Emission
Excitation
Abs max: 550 nm Em max: 568 nm
550
650
Wavelength (nm)
DyLight 549
500
800
Abs max: 682 nm Em max: 715 nm
Wavelength (nm)
450
750
Emission
Emission 470
700
DyLight 488
Excitation
420
650
Wavelength (nm)
Abs max: 493 nm Em max: 518 nm
370
600
Wavelength (nm)
Abs max: 770 nm Em max: 794 nm
650
700
700
Emission
350
Excitation
300
Abs max: 646 nm Em max: 674 nm
Excitation
Emission
Excitation
Abs max: 400 nm Em max: 420 nm
Emission
DyLight 649
750
800
Wavelength (nm)
Wavelength (nm)
DyLight 633
Emission
Excitation
Abs max: 638 nm Em max: 658 nm
500
550
600
650
700
750
Wavelength (nm)
Figure 1. Excitation and emission spectra for Thermo Scientific DyLight Dyes.
5
Dyes (Cont.)
Ordering Information Product # Description Amine-Reactive Dyes 46400 DyLight 405 NHS Ester 46401 DyLight 405 NHS Ester 46402 DyLight 488 NHS Ester 46403 DyLight 488 NHS Ester 46407 DyLight 549 NHS Ester 46408 DyLight 549 NHS Ester 46414 DyLight 633 NHS Ester 46417 DyLight 633 NHS Ester 46415 DyLight 649 NHS Ester 46416 DyLight 649 NHS Ester 46418 DyLight 680 NHS Ester 46419 DyLight 680 NHS Ester 46421 DyLight 800 NHS Ester 46422 DyLight 800 NHS Ester Sulfhydryl-Reactive Dyes 46660 DyLight 405 Maleimide 46602 DyLight 488 Maleimide 46607 DyLight 549 Maleimide 46613 DyLight 633 Maleimide 46615 DyLight 649 Maleimide 46618 DyLight 680 Maleimide 46621 DyLight 800 Maleimide
6
Pkg. Size
U.S. Price
1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg 1 mg 5 x 50 µg
$199 $159 $199 $159 $199 $159 $199 $159 $199 $159 $199 $159 $199 $159
1 mg 1 mg 1 mg 1 mg 1 mg 1 mg 1 mg
$199 $199 $199 $199 $199 $199 $199
Dye Removal Columns
High-performance dye removal columns Convenient new options for fluorescent antibody labeling The same high performance dye removal spin-columns used in the Thermo Scientific DyLight Antibody Labeling Kits are now available separately.
Highlights: • Fast – remove free dye from conjugates in approximately 30 seconds • Efficient – purification resin provides outstanding conjugate recovery (Figure 1) • Minimal sample dilution
100%
Conjugate Recovery
80%
60%
40%
85%
84%
84%
(n = 12)
(n = 12)
(n = 12)
FITC 79% 90% 85%
Fluorescein 86% 81% 84%
Rhodamine 94% 74% 84%
20%
0%
Conjugate Goat anti-mouse IgG Streptavidin Average Recovery
Figure 1. Thermo Scientific DyLight Dye Removal Columns provide outstanding conjugate recovery.
Ordering Information Product # 22858
Description Dye Removal Columns
Pkg. Size Kit
Includes: Purification Resin Spin Columns Microcentrifuge Collection Tubes
5 ml 10 ea. 20 ea.
U.S. Price $ 99
7
Antibody Labeling Kits
Label and purify antibodies in one hour Antibody labeling kits for fast and efficient labeling and exceptional recovery The Thermo Scientific DyLight Antibody Labeling Kits were specifically developed for fast, efficient labeling of antibodies. Two convenient kit formats are available to accommodate varied labeling requirements. The Antibody Labeling Kits contain all necessary components to perform three separate labeling reactions using 1 mg of IgG or similar quantities of other proteins. The DyLight Microscale Antibody Labeling Kits contain all the necessary components to perform five separate labeling reactions using 100 µg of IgG. The labeling kits use high-performance spin desalting columns to provide exceptional dye removal and antibody recovery (Figure 1).
Highlights: • Fast – fluorescently label and purify protein in approximately one hour • Amine-reactive dyes – label virtually any protein • Pre-measured fluorescent dye – eliminate the time, waste and hassle associated with weighing dye
• Efficient non-reacted dye removal • Minimal sample dilution • Spin column format eliminates the need for column preparation, fraction screening and waiting for protein to emerge from column • Easy protocol (Figure 2)
Antibody Recovery
100
Figure 1. Thermo Scientific DyLight Antibody Labeling Kits provide outstanding recovery. The percent recovery for DyLight Antibody Labeling Kits is the average for 16 labeling reactions using three different antibodies. The percent recovery for DyLight Microscale Antibody Labeling Kits is the average for 13 labeling reactions using three different antibodies.
80 60 40
81%
75%
Thermo Scientific DyLight Antibody Labeling Kit
Thermo Scientific DyLight Microscale Antibody Labeling Kit
20 0
Table 1. Spectral properties of Thermo Scientific DyLight Fluorescent Dyes. Emission Blue Green Yellow Red Red Near Infrared
DyLight Dye 405 488 549 633 649 680 800
Ex/Em* 400/420 493/518 550/568 638/658 646/674 682/715 770/794
*Excitation and emission maxima in nanometers †Molar extinction coefficient (M-1 cm-1)
8
ε† 30,000 70,000 150,000 170,000 250,000 140,000 270,000
Spectrally Similar Dyes Alexa 405 and Cascade Blue Alexa 488, fluorescein and FITC Alexa 546, Alexa 555, Cy3 and TRITC Alexa 633 Alexa 647 and Cy5 Alexa 680 and Cy5.5 IRDye 800
Step 1. Labeling reaction
Step 2. Removal of excess fluorescent dye
Centrifuge 30 seconds
Add antibody to vial containing pre-measured dye. Incubate 1 hour at room temperature.
Apply labeling reaction to Spin Desalting Column.
Recover labeled antibody.
Figure 2. Protocol summary for Thermo Scientific DyLight Antibody Labeling Kits.
Microscale Kits Contain sufficient reagents to label and purify 5 x 100 µg of IgG In addition to contents listed below, all Microscale Kits include: • Reaction Buffer, 1 ml • Microcentrifuge Collection Tubes, 10 each • Spin Columns, 5 each • Purification Resin, 5 ml
Ordering Information Product # 53021
Description DyLight 405 Microscale Antibody Labeling Kit
Pkg. Size Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials Kit 5 vials
DyLight 405 NHS Ester
53025
DyLight 488 Microscale Antibody Labeling Kit DyLight 488 NHS Ester
53035
DyLight 549 Microscale Antibody Labeling Kit DyLight 549 NHS Ester
53047
DyLight 633 Microscale Antibody Labeling Kit DyLight 633 NHS Ester
53051
DyLight 649 Microscale Antibody Labeling Kit DyLight 649 NHS Ester
53057
DyLight 680 Microscale Antibody Labeling Kit DyLight 680 NHS Ester
53063
DyLight 800 Microscale Antibody Labeling Kit DyLight 800 NHS Ester
U.S. Price $275 $275 $275 $275 $275 $275 $275
Antibody Labeling Kits Contain sufficient reagents to label and purify 3 x 1 mg of IgG or similar quantities of other proteins In addition to contents listed below, all Antibody Labeling Kits include: • Reaction Buffer, 1 ml • Spin Columns, 6 each
• Microcentrifuge Collection Tubes, 12 each • Purification Resin, 5 ml
Ordering Information Product # 53020
Description DyLight 405 Antibody Labeling Kit DyLight 405 NHS Ester
53024
DyLight 488 Antibody Labeling Kit DyLight 488 NHS Ester
53034
DyLight 549 Antibody Labeling Kit DyLight 549 NHS Ester
53046
DyLight 633 Microscale Antibody Labeling Kit DyLight 633 NHS Ester
53050
DyLight 649 Antibody Labeling Kit DyLight 649 NHS Ester
53056
DyLight 680 Antibody Labeling Kit DyLight 680 NHS Ester
53062
DyLight 800 Antibody Labeling Kit DyLight 800 NHS Ester
Pkg. Size Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials Kit 3 vials
U.S. Price $275 $275 $275 $275 $275 $275 $275
9
Conjugates
Thermo Scientific DyLight Conjugates Excellent brightness make these conjugates a clear alternative
Highlights: • Available conjugated to commonly used secondary antibodies, streptavidin and NeutrAvidin Biotin-Binding Protein • Molar ratio (dye:protein) optimized to provide excellent fluorescent intensity • Stable for 1 year at 4°C • Antibody conjugates are affinity-purified to minimize cross-reactivity
Western Blotting Figure 1. Two-color infrared Western blot detection of p53 and cyclophilin B knockdown using Thermo Scientific DyLight 680- and Thermo Scientific DyLight 800-labeled secondary antibodies. Protein lysate from transfected A549 cells was separated using SDS-PAGE and transferred to PVDF membrane. Lane 1: MW marker, Lane 2: mock transfected sample, Lane 3: negative control siRNA, Lane 4: siRNA targeted against p53 and Lane 5: siRNA targeted against cyclophilin. The membranes were imaged with the Odyssey Infrared Imaging System using the 700 and 800 channels.
1
10
2 3
4
5
Microplate-Based Assays
Relative Fluorescent Intensity
60,000
Figure 2. Thermo Scientific DyLight 649 Streptavidin and Thermo Scientific NeutrAvidin Conjugates are brighter than Alexa 647 Streptavidin Conjugates in microplate-based assays. Microplates were coated with biotinylated BSA at the indicated concentrations. DyLight 649 and Alexa 647 Conjugates were diluted 1:250 (from 1 mg/ml stock solutions) in PBS and 100 µl was applied to each plate. Fluorescent intensity was measured with a Tecan Safire™ Microplate Reader using Cy5 Dye settings.
50,000 40,000 30,000
20,000 DyLight 649 Streptavidin DyLight 649 NeutrAvidin Alexa 647 Streptavidin
10,000 0 0
50
100
150
200
250
Biotinylated BSA (ng/well)
Relative Fluorescent Intensity
50,000
Figure 3. Thermo Scientific DyLight 549 Goat AntiMouse Conjugates are brighter than Alexa 555 Dye conjugates in microplate-based assays. Microplates were coated with Mouse IgG at the indicated concentrations. DyLight 549 and Alexa 555 Conjugates were diluted 1:250 (from 1 mg/ml stock solutions) in PBS and 100 µl was applied to each plate. Fluorescent intensity was measured with a Tecan Safire Microplate Reader using Cy3 Dye settings.
45,000 40,000 35,000 30,000 25,000 20,000 15,000 DyLight 549 Goat anti-mouse IgG
10,000
Alexa 555 Goat anti-mouse IgG
5,000 0
0
200
400
600
800
1,000
1,200
Mouse IgG (ng/well)
Relative Fluorescent Intensity
16,000,000
Figure 4. Thermo Scientific DyLight 680 and Thermo Scientific DyLight 800 Streptavidin Conjugates are brighter than Alexa 680 or IRDye 800 Conjugates in microplate-based assays. Microplates were coated with biotinylated BSA at the indicated concentrations. Conjugates were diluted to 10 µg/ml in PBS and 100 µl was applied to each plate. Fluorescent intensity was measured with the Odyssey Infrared Imaging System using the 700 and 800 channels.
DyLight 800 Streptavidin IRDye 800 Streptavidin 12,000,000 DyLight 680 Streptavidin Alexa 680 Streptavidin
8,000,000
4,000,000
0
2
4 6 8 Biotinylated BSA (ng per well)
10
12
Ordering Information Conjugates: Package size for these items is 1 mg at 1 mg/ml. Product # / U.S. Price DyLight DyLight DyLight Description 488 Dye 549 Dye 649 Dye Goat Anti-Mouse 35502/$128 35507/$128 35515/$128 IgG (H+L) Goat Anti-Rabbit 35552/$128 35557/$128 35565/$128 IgG (H+L) Streptavidin 21832/$155 21837/$155 21845/$155 NeutrAvidin 22832/$170 22837/$170 22845/$170 Biotin-Binding Protein
DyLight DyLight 680 Dye 800 Dye 35518/$128 35521/$128 35568/$128 35571/$128 21848/$155 21851/$155 22848/$170 22853/$170
11
Fluorescent Protein Molecular Weight Markers
Fluorescent MW markers for Western blotting and in-gel detection One- or two-color fluorescence detection with one protein molecular weight marker Fluorescent Thermo Scientific DyLight Infrared Protein Molecular Weight Markers have been optimized for direct visualization of marker proteins after SDS-polyacrylamide gel electrophoresis (PAGE) (Figure 1). Two convenient formats are available to accommodate fluorescence imaging requirements in the visible or near-infrared region of the spectrum. Each protein in the mixture is labeled with two fluorescent dyes to provide flexible one- or two-color detection with common fluorescence imaging systems (Table 1). The markers are compatible with Western blotting (Figure 2) and can be detected by virtually any in-gel staining method (Figure 3). The DyLight Protein Molecular Weight Markers consist of nine proteins with molecular weights in the range of 6K to 200K.
Highlights: • Easily multiplexed – two excitation and emission maxima enable one- or two-color fluorescence detection • Saves time – eliminate the need for awkward marking or overlay procedures • Fluorescence and colorimetric – two detection options available: in-gel or on-membrane
• Instrument-compatible – spectra are compatible with common fluorescence imaging systems • Photostable – capture multiple images with no decrease in fluorescent intensity
Table 1. Spectral characteristics of Thermo Scientific DyLight Fluorescent Protein Molecular Weight Markers.
DyLight 547 Fluors DyLight 647 Fluors
12
Excitation (nm) 557 652
Emission (nm) 574 673
Extinction Coefficient (min) 150,000 M-1 cm-1 250,000 M-1 cm-1
Figure 1 A B
Figure 2 A B
Figure 3 A B
Myosin (200K) Phosphoylase B (97K) BSA (66K) Protein A (42K) Protein L (36K) Peanut Agglutinin (28K) Trypsin Inhibitor (20K) Lysozyme (14K) Aprotinin (6K)
Fluorescent and Colorimetric Detection of Thermo Scientific DyLight Fluorescent Protein Molecular Weight Markers Figure 1. Panel A. Direct in-gel fluorescent detection. Marker proteins (5 µl) were separated in 4-20% Tris-glycine gels. The gels were imaged with the Kodak Image Station 2000MM using a five-minute exposure at f2.8 with the A. 535/600 nm excitation/emission filter set and B. 625/700 nm excitation/emission filter set. Figure 2. Fluorescent Western blot detection. Marker proteins (5 µl) were separated in 4-20% Tris-glycine gels and transferred to A. nitrocellulose or B. PVDF membrane. Blots were imaged with the Typhoon 9410 at 500V PMT using the A. Cy3 Fluor and B. Cy5 Fluor laser settings. Note: Proteins in the marker mix produce uniform fluorescent intensities in SDS-PAGE applications; however, variations in protein-transfer efficiency affect fluorescent intensity. For example, high molecular weight proteins, such as myosin (200K), typically transfer less efficiently than low molecular weight proteins. Figure 3. Colorimetric in-gel detection. Marker proteins (10 µl) were separated in 4-20% Tris-glycine gels and stained with A. Thermo Scientific Imperial Protein Stain† and B. silver stain.
Ordering Information Product # 22859
Description DyLight Infrared Protein Molecular Weight Markers
Pkg. Size 250 µl
U.S. Price $199
250 µl
$199
Sufficient material for loading 50 gel lanes
26665
DyLight Fluorescent Protein Molecular Weight Markers
Sufficient material for loading 50 gel lanes. † See patent information on inside back cover
13
Western Blotting Kits
Multiplex Fluorescent Western Blotting Kits Fluorescent Western blotting made easy The fluorescence detection of two different targets on a single Western blot is now easier to perform with the new Thermo Scientific DyLight 680/800 and 549/649 Western Blotting Kits. The kit provides a highly optimized and convenient format to save you the time and frustration of having to evaluate reagents for compatibility with fluorescent Western blotting (Figure 1). Each kit contains sufficient reagents for 10 Western blots and includes the new Thermo Scientific Pierce Dual-Labeled Protein Molecular Weight Markers† and secondary antibodies conjugated to DyLight 680 and 800 Fluorescent Dyes (Table 1). The DyLight 680/800 Western Blotting Kit uses DyLight 680 and DyLight 800 labeled secondary antibodies for imaging in the near-infrared region (e.g. LI-COR Odyssey Imaging System) of the spectrum (Figure 2). Highlights: • Easily multiplexed – detect two different targets on a single Western blot • Optimized format – protocol requires little to no optimization, saving time and the frustration of having to evaluate and optimize reagents (e.g., secondary antibody dilutions, etc.) • Plug-and-play – kits include DyLight-labeled secondary antibodies, blocking buffer, wash buffer, dual-labeled fluorescent molecular weight markers and low-fluorescence membranes
• Instrument-compatible – DyLight Fluor spectra match the principal output wavelengths of common fluorescence instrumentation • Optimized format – provides low background and high signal intensity • Convenient – saves time and money associated with optimizing fluorescent Western blots
Table 1. Spectral properties of Thermo Scientific DyLight Western Blotting Kits.
12 ng
6 ng
3 ng
1 ng
0.5 ng
12 ng 6 ng 3 ng 1 ng
25 ng
*Excitation and emission maxima in nanometers †Molar extinction coefficient (M-1 cm-1)
Ex/Em*
ε†
Spectrally Similar Dyes
550/568 646/674
150,000 250,000
Alexa 546, Alexa 555, Cy3 and TRITC Alexa 647 and Cy5
682/715 770/794
140,000 270,000
Alexa 680 and Cy5.5 IRDye 800
25 ng MW Marker
Western Blotting Kit DyLight 549/649 Western Blotting Kit DyLight 549 Goat Anti-Mouse IgG (H+L) DyLight 649 Goat Anti-Rabbit IgG (H+L) DyLight 680/800 Western Blotting Kit DyLight 680 Goat Anti-Mouse IgG (H+L) DyLight 800 Goat Anti-Rabbit IgG (H+L)
Tubulin
25 ng
12 ng
6 ng
3 ng
1 ng
0.5 ng
12 ng 6 ng 3 ng 1 ng
25 ng
ECL Plex Western Blotting Kit
Tubulin
TNFα
14
DyLight 549/649 Western Blotting Kit
MW Marker
TNFα
Figure 1. The Thermo Scientific DyLight 549/649 Western Blotting Kit provides lower background and higher signal in two-color Western blot detection compared to a competing fluorescent Western blotting kit. Proteins were separated in 4-20% Precise Protein Gels and transferred to low-fluorescence PVDF membrane. The membranes were blocked overnight in 1% BSA and target proteins were detected following manufacturer-recommended protocols. Membranes were imaged with the Typhoon 9410.
50 µg
25 µg
12.5 µg
6.25 µg
0.063 µg
0.125 µg
0.25 µg
0.5 µg
1 µg
Figure 2. The Thermo Scientific DyLight 680/800 Western Blotting Kit provides low background and high signal in two-color Western blot detection. Proteins were separated in 4-20% Thermo Scientific Precise Protein Gels and transferred to LowFluorescence PVDF Transfer Membrane (Product # 22860). The membrane was blocked overnight in SEA BLOCK Blocking Buffer (Product # 37527) and target proteins were detected following the recommended protocol. Membranes were imaged with the LI-COR Odyssey Infrared Imaging System. Tubulin was detected from the indicated quantity of HeLa cell lysate. Purified TNFα was detected at the indicated quantity.
Tubulin
TNFα
Ordering Information Product # 22855
22854
Description DyLight 680/800 Western Blotting Kit
Pkg. Size Kit
Sufficient reagents for 10 Western blots. Includes: DyLight 680 Goat Anti-Mouse IgG (H+L) DyLight 800 Goat Anti-Rabbit IgG (H+L) DyLight Infrared Protein Molecular Weight Markers Wash Buffer (30X) SEA BLOCK Blocking Buffer Low-Fluorescence PVDF Transfer Membrane
120 µl 120 µl 30 µl 200 ml 500 ml 10 ea.
DyLight 549/649 Western Blotting Kit
Kit
Sufficient reagents for 10 Western blots. Includes: DyLight 549 Goat Anti-Mouse IgG (H+L) DyLight 649 Goat Anti-Rabbit IgG (H+L) Fluorescent Dual-labeled Protein MW Markers Wash Buffer (30X) Blocker BSA in PBS (10X) Low-Fluorescence PVDF Transfer Membrane
50 µl 50 µl 60 µl 200 ml 50 ml 10 ea.
Complementary Products Product # Description 22859 DyLight Infrared Protein Molecular Weight Markers
U.S. Price $199
$199
Pkg. Size 250 µl
U.S. Price $199
10/pkg.
$ 78
250 µl 500 ml Kit
$199 $129 $275
Kit
$275
Kit
$275
Kit
$275
Sufficient material for loading 50 gel lanes.
22860 26665 37527 53056
Low-Fluorescence PVDF Transfer Membrane, 0.2 µm 7 cm x 8.4 cm DyLight Dual Fluor-Labeled Protein MW Markers SEA BLOCK Blocking Buffer DyLight 680 Antibody Labeling Kit Sufficient reagents to label and purify 3 x 1 mg of IgG
53057
DyLight 680 Microscale Antibody Labeling Sufficient reagents to label and purify 5 x 100 µg of IgG.
53062
DyLight 800 Antibody Labeling Kit Sufficient reagents to label and purify 3 x 1 mg of IgG.
53063
DyLight 800 Microscale Antibody Labeling Kit
Sufficient reagents to label and purify 5 x 100 µg of IgG. † See patent information on inside back cover
15
Fluorescent Dye-Based Stains
Thermo Scientific Krypton Fluorescent Protein Stain A faster, affordable fluorescent stain that provides excellent performance Thermo Scientific Krypton Protein Stain† is a fluorescent stain for detecting proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D gels.
Highlights • Excitation/emission maxima – 520/580 nm • Compatibility – works with all SDS-polyacrylamide and 2-D gel types and with MS analysis • Linear quantitative range – three to four orders of magnitude • Sensitive – detects down to 0.25 ng protein with the basic 2.7-hour protocol • Fast – using the rapid protocol, detects down to 2 ng protein in 30 minutes • Comparative – minimal differential staining of proteins
Ordering Information Product # 46628
Description Krypton Protein Stain (10X)
Pkg. Size 20 ml
U.S. Price $ 39
100 ml
$110
500 ml
$399
Sufficient reagent to stain four mini gels (8 cm x 10 cm)
46629
Krypton Protein Stain (10X) Sufficient reagent to stain 20 mini gels (8 cm x 10 cm) or two to four large-format gels
46630
Krypton Protein Stain (10X)
Sufficient reagent to stain 100 mini gels (8 cm x 10 cm) or 10 to 20 large-format gels † See patent information on inside back cover
16
www.thermo.com/dylight
Pierce Protein Research Products Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316 • Customer Assistance E-mail: [email protected] Outside the United States, visit our website or call 815-968-0747 to locate your local branch office or distributor. © 2007 Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Prices listed herein were accurate at the time of printing. Visit our website for up-to-date prices. † U.S. patent pending on Dual-labeled Fluorescent Molecular Weight Marker Technology. U.S. patent pending on Imperial Protein Stain Technology. U.S. patent pending on Krypton Infrared Protein Stain Technology. Alexa® is a trademark of Molecular Probes, Inc. CyDye®, Cy® and Typhoon® are trademarks of GE Healthcare. Odyssey® is a trademark of LI-COR Biosciences. All other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries.
19
1601552 11/07
61105-0117
PO Box 117
• High water solubility • Excellent photostability
Thermo Scientific DyLight Fluors
Rockford, IL
Pierce Protein Research Products
MADISON, WI PERMIT NO. 846
PAID
PRSRTD STD U.S. POSTAGE
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