Two-dimensional Polyacrylamide Gel Electrophoresis

Two-Dimensional Polyacrylamide Gel Electrophoresis Author: G.D. Heda. Asst Professor of Genetics, The University of Tennessee Health Sciences Center, Research Biologist, V.A. Medical Center, 1030 Jefferson Avenue, Memphis, Tennessee 38104, USA (901) 577-7269 – Office e-mail: [email protected] INTRODUCTION Two-dimenstional Polyacrylamide Gel Electrophoresis (2-D PAGE) is commonly used to separate complex mixture of proteins based on their iso-electric point (pI), and molecular weight (MW). (a) First Dimension: The proteins are first separted on the basis of their pI. The pI is defined as the pH of a protein at which a protein carries no net charge and will not further migrate in an electric field. Although pI values usually fall in the range of pH 3-12, with the majority of proteins falling between pH 4 and pH 7. This technique of separating proteins based on their pI is called isoelectric focusing (IEF). IEF was traditionally performed on a tube gel containing ampholyte-based self-forming internal gradients. The current protocol is based on the use of commercially abvailable flat bed immobilized pH gradient (IPG) srips of pH range 3-10. (BioRad Laboratories, Hercules,CA). (b) Second Dimenstion: Proteins immobilized on IPG strips are than placed on a vertical SDS-PAGE slab gel and electrophoresed to separate proteins on the basis of their molecular weight. 2-D PAGE, lately, has gained special importance with the evolving new science of `Proteomics’. Proteome analysis is the direct measurement of proteins in a given sample at a given time. 2-D PAGE allows analysis and identification of hundreds of thousands of gene products with computer assisted software programs and rapidly growing protein data-bases. BUFFERS REHYDRATION/SAMPLE BUFFER Urea (F.W. 60.06) 10.5 g (7M) Thiourea 3.8 g (2M) CHAPS 1 g (4%) DTT (F.W. 76.12)* 232.5 mg (60 mM) 100x Bio-Lyte 3/10 ampholyte 125 µl (0.5%) (Bio-Rad # 163-2094) 0.1% Bromophenol Blue^ 250 µl (0.001%) H2O To make 25 ml final G. Heda: 1/6 2D -Gel Electrophoresis Provided through: The European Working Group on CFTR Expression

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Prepare and store as 0.5-1 ml aliquots at –20oC (without DTT, BPB). Add * (DTT) as 9.3 mg/ml just before use. Add this buffer directly to purified protein pellet. Remove 5-10 µl for protein assay and add ^ (BPB), as 25 µl/ml. EQUILIBRATION BUFFER Urea (F.W. 60.06) SDS 1.5 M Tris-HCl (pH8.8) 50% Glycerol H2O

36 g (6M) 2 g (2%) 3.3 ml (50 mM) 60 ml (30%) To make 100 ml final

Freeze buffer in 10 ml aliquots. • •

Add 2% DTT (200 mg/10 ml) to make Equilibration Buffer-I, or Add 2.5% Iodoacetamide (250 mg/10 ml) to make Equilibration Buffer-II

OVERLAY AGAROSE Low-melt agarose 1x Tris-Glycine-SDS 1% Bromophenol Blue

0.5 g (0.5%) 100 ml 100 µl of 1% (w/v) stock solution (0.001%)

ELECTRODE BUFFER Glycine Tris 10% SDS Water to make

57.7 g 12.1 g 45 ml 4 l ml

IEF STAINING SOLUTION Coomassie brilliant blue R Methanol Acetic acid Water

0.5 g (0.025%) 800 ml (40%) 140 ml (7%) 1060 ml

IEF DE-STAINING SOLUTION Coomassie brilliant blue R Methanol Acetic acid Water

0.5 g (0.025%) 100 ml (5%) 140 ml (7%) 1760 ml

2-D PAGE STAINING SOLUTION GelCode Blue Stain Reagent (Pierce # 24590)

G. Heda: 2/6 2D -Gel Electrophoresis Provided through: The European Working Group on CFTR Expression

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GEL DRYING SOLUTION (Pre-soaking Buffer) Glycerol 100 ml (10%) Ethanol 200 ml (20%) Water 700 ml PROCEDURE Day-1 (A) Iso-Electric Focussing (IEF) 1. Purify protein samples (300-500 µg) using PlusOne 2-D Clean-Up Kit (Amersham # 80-6484-51), as per the manufacturer’s protocol. 2. Re-suspend the protein pellet in 185 µl of Re-hydration/Sample Buffer. Make sure that the sample is clear. If necessary quickly sonicate once on ice followed by quick spin of samples on a centrifuge. 3. Re-hydrate an 11-cm long IPG strip (pH 3-10) with the protein sample on a disposable re-hydration/equilibration tray for overnight at the room temperature. Overlay each strip with 2-3 ml of mineral oil to prevent evaporation during the rehydration process. Note: Set the tray on a level bench. Note: Protein measurement in samples re-suspended in sample buffer (except BPB) can be performed by using Bio-Rad DC Protein Assay kit (Bio-Rad # 500-0112). Day-2 4. Place Electrode Wicks (Bio-Rad # 1654071) on anode and cathode ends of electrode wires on an IEF tray. Wet the wicks by adding 9 µl of nano-pure water. 5. Remove IPG strip, drain oil, and lay it over on wet electrode wicks. Overlay each strip with 2-3 ml of mineral oil to prevent evaporation during IEF process. 6. Start IEF on Protean IEF cell (Bio-Rad # 165-4000) at 20ºC using the following step cycles: (a) Step 1 250 volts, 20 min, linear ramp (b) Step 2 8000 volts, 2.5 hr, linear ramp (c) Step 3 8000 volts, 20,000 V-hr, rapid ramp Total: ~30,000 V-hr for a period of 5.3 hr After IEF, remove IPG strips, drain oil and either proceed with 2nd dimension or freeze at –70ºC. Staining IPG Strips Drain oil off the IPG strips. Place a wet whatman-1 filter paper gently over the gel surface, to remove oil as much as possible. Stain with IEF gel staining solution for 30-60 minutes. Destain until bands are visible. G. Heda: 3/6 2D -Gel Electrophoresis Provided through: The European Working Group on CFTR Expression

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Day-3 2nd Dimension Gel Electrophoresis 7. Thaw IPG strips at room temperature for 10-15 minutes. Equilibrate with 3-4 ml/strip of Equilibration Buffer-I, followed by Equilibration Buffer-II for 10 minutes each. 8. Dip IPG strip in Electrode Buffer and place it on a 4-20% gradient SDS-PAGE. Electrophorese at 200 volts using Criterion Dodeca Cell (Bio-Rad # 165-4130) attached to a cooling water bath circulator. Staining of 2nd dimension gel 9. Coomassiee-Colloidal stain: a) Remove gel and wash 5x with 100 ml of distilled water for 10 minutes each time. b) Stain with 20 ml of Pierce GelCode Blue Stain for 1 hour. For overnight stain add 2 ml of 20%NaCl (w/v) in water for every 20 ml of stain. Note: Mix the stain well before use. c) Destain the gel with 100 ml of distilled water for 1 hour. The gel can be left in water for several days without any loss of sensitivity. 10. Silver stain: a) Fixation: • Fix gel in 50% methanol, 5% Acetic acid for 20 minutes. • Wash in 50% methanol for 10 mintues. • Wash in water for 10 minutes (to remove remaining acid). b) Sensitization: • Incubate with 0.02% sodium thiosulfate for 1 minute • Rinse twice with water for 1 minute each. c) Silver Reaction: • Submerge gel in chilled 0.1% silver nitrate for 20 minutes at 4oC. • Rinse twice with water for 1 minute each. d) Developing: • Place gel in 2% sodium carbonate containing 0.04% formalin (add formalin just before use; formalin = 35% formaldehyde in water) • Swirl until desired intensity of staining occurs. • If developer turns yellow, then discard and replace with fresh stuff e) Stopping: • Wash the gel in 5% acetic acid for 10 minutes. f) Storage: • Store in 1% acetic acid at 4oC. g) Scan the wet or dried gel using a scanner at 300 dpi resolution.

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Note: A variety of silver staining protocols is available with slight variations in fixing and developing. The author has found the above protocol as suitable in terms of acceptable background and bands sensitivity. This is an EMBL approved protocol. GEL DRYING • •

Soak the gel for 30 minutes in Pre-soaking buffer. Sandwich the gel between the two layers of cellophane avoiding air-bubbles against a solid plate. Air dry for overnight.

ANALYSIS Analyze digital gel images in a TIFF format using PDQuest software (Bio-Rad #1708611). REFERENCES 1. O’Farrell, P.H. (1975). High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021. 2. Protein Electrophoresis – Applications Guide (1994). Hoefer Scientific Instruments, San Francisco, CA. 3. 2-D Electrophoresis for Proteomics – A Method and Product Manual. Bulletin 2651. Bio-Rad Laboraroties, CA. 4. Moertz. E., Krogh. T.N., Vorum, H., Angelika Gorg. Improved silver staining protocols compatible with large-scale protein identification. [http://www.protana.com/PDF/ASMS/PosterSilverstain.pdf]

Note: Design of this protocol is based on the reagents and supplies mostly obtained commercially from Bio-Rad Laboratories. However, in no-way this is meant to advertise or solicit Bio-Rad’s products.

G. Heda: 5/6 2D -Gel Electrophoresis Provided through: The European Working Group on CFTR Expression

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A

B Fig 1 - 2-D electrophoretic profiles of CFTR enriched vesicles from LLC-PK1 (wild-type) cells. A - Complete view; B - Magnified view. Boxed magnification in lower panel (B) is provided for clear view of protein spotting patterns. pI locations on the gel were marked by super-imposing a 2-D gel containing 2-D SDS-PAGE standards (Bio-Rad # 1610320). Purified vesicle enriched fraction was re-suspended in 180 l of Sample buffer and applied to a 11 cm long IPG strip (pH3-10). Second dimension gel was performed on a 4-20% gradient SDS-PAGE.

G. Heda: 6/6 2D -Gel Electrophoresis Provided through: The European Working Group on CFTR Expression

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