Two-Dimensional Polyacrylamide Gel Electrophoresis by murplelake80

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									           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: gheda@utmem.edu

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                                    14-03-2003
2D -Gel Electrophoresis
            Provided through: The European Working Group on CFTR Expression
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)                                  36 g (6M)
SDS                                                2 g (2%)
1.5 M Tris-HCl (pH8.8)                          3.3 ml (50 mM)
50% Glycerol                                       60 ml (30%)
H2O                                           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                                       0.5 g (0.5%)
1x Tris-Glycine-SDS                                      100 ml
1% Bromophenol Blue                     100 µl of 1% (w/v) stock solution (0.001%)

ELECTRODE BUFFER
Glycine                                               57.7 g
Tris                                                  12.1 g
10% SDS                                               45 ml
Water to make                                         4 l ml

IEF STAINING SOLUTION
Coomassie brilliant blue R                       0.5 g (0.025%)
Methanol                                          800 ml (40%)
Acetic acid                                        140 ml (7%)
Water                                                1060 ml

IEF DE-STAINING SOLUTION
Coomassie brilliant blue R                       0.5 g (0.025%)
Methanol                                          100 ml (5%)
Acetic acid                                       140 ml (7%)
Water                                               1760 ml

2-D PAGE STAINING SOLUTION

GelCode Blue Stain Reagent (Pierce # 24590)




G. Heda:                                   2/6                                  14-03-2003
2D -Gel Electrophoresis
            Provided through: The European Working Group on CFTR Expression
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                                 14-03-2003
2D -Gel Electrophoresis
            Provided through: The European Working Group on CFTR Expression
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.



G. Heda:                                   4/6                                14-03-2003
2D -Gel Electrophoresis
            Provided through: The European Working Group on CFTR Expression
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 #170-
8611).

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                                     14-03-2003
2D -Gel Electrophoresis
            Provided through: The European Working Group on CFTR Expression
                                              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 # 161-
0320). 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                                 14-03-2003
2D -Gel Electrophoresis
            Provided through: The European Working Group on CFTR Expression

								
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