Protocols

Two-Dimensional Gel Electrophoresis - From Start to Finish

Posted on Thursday, August 24, 2006

Description
Two-dimensional gel electrophoresis separate proteins based on their charge [isoelectric point (pI)] and molecular mass. This technique has become an important tool in new emerging science of `Proteomics', with rapidly growing volume of sequence data produced by the genome projects and with advancement of new mass spectrometry methods. The described protocol is standarized with the first and second dimensional gel apparatus of Bio-Rad Laboratories.

Procedure
(A) ISO-ELECTRIC FOCUSSING (IEF):

DAY-1

1. Purify protein samples (300-500 ?g) using PlusOne? 2-D Clean-Up Kit (Amersham Cat.# 80-6484-51).
2. Resuspend the pellet in 185 ?l of Re-hydration/Sample Buffer. Make sure that the sample is clear. If necessary quickly sonicate once on ice and quick spin.
3. Re-hydrate 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.

DAY-2

4. Place Electrode Wicks (BioRad Cat. # 1654071) on anode and cathode ends of electrode wires on an IEF tray. Wet the wicks by adding 9 ?l 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 (BioRad Cat. # 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
(d) Step 4 500 volts, 99 hrs (hold ramp)
Total: ~30,000 V-hr for a period of 5.3 hr

Following 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 (BioRad Cat. # 161-0434) for 30-60 minutes. Destain (Bio-Rad Cat. # 161-0438).

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.

CAUTION: IPG strips must remain in Equi. Buffer-II, until ready to be loaded on 2nd dimension gel. Keeping them in Electrode Buffer will cause hydration of acrylamide gel of IPG strip and smearing in 2nd dimension gel.

8. Dip IPG strip in Electrode Buffer and place it on a 4-20% gradient SDS-PAGE. Seal the IPG strips using molten overlay agarose, cooled down to 40-50oC. Make sure that there are no air-bubbles between IPG strip and slab gel and on the back of IPG strip.

9. Electrophorese at 200 volts using Criterion� Dodeca�? Cell (BioRad Cat.# 165-4130) attached to a cooling water bath circulator.

STAINING:
(A) COOMASSIE STAINING:

1. Remove gel and wash 3x with 100 ml each of distilled water.

2. Stain with 20 ml of Pierce GelCode Blue Stain (Pierce Cat. # 24590) or Simple Blue Safe Stain (Invitrogen Cat. #46-5034) for 1 hour. For overnight stain add 2 ml of 20% NaCl (w/v) in water for every 20 ml of stain.

3. Destain with 100 ml of distilled water for 1 hr. The gel can be left in water for several days without loss of sensitivity.

(B) SILVER STAINING (EMBL protocol):

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).
(Addl. washing overnight willl reduce background staining).
b) Sensitization:
� Incubate with 0.02% sodium thiosulfate for 1 minute
(Prepare fresh: 50 mg/250 ml H2O)
� Rinse twice with water for 1 minute each.
c) Silver Reaction:
� Submerge gel in chilled 0.1% silver nitrate for 20 minutes at 4oC.
(Prepare fresh: 250 mg/250 ml H2O)
� Rinse twice with water for 1 minute each.
d) Developing:
� Place gel in 2% sodium carbonate containing 0.04% formalin.
(Prepare fresh: 5 g sodium carbonate/250 ml H2O + 97 ?l of 37% formaldehyde. Do not add formaldehyde until ready)
� Swirl until desired intensity of staining occurs.
� If developer turns yellow, then discard and replace with fresh solution.
e) Stopping:
� Wash the gel in 5% acetic acid for 10 minutes.
f) Storage:
� Store in 1% acetic acid at 4oC.

Scan the wet or dried stained gels using a scanner at 300 dpi resolution.

GEL DRYING:

1. Soak the gel for 30 minutes in Gel Drying Solution.
2. Sandwich the gel between the two layers of cellophane avoiding air-bubbles against a solid plate. Air dry for overnight.
3. Gels can also be dried using commercially available gel driers using vacuum and hot plate. However, this type of devices are not good for gradient gels, and may cause cracks during the drying process.

ANALYSIS:

Analyze digital gel images in a TIFF format using PDQuest� software (BioRad Cat. #170-8611).


Recipes
1. REHYDRATION/SAMPLE BUFFER

Urea (F.W. 60.06) --- --- 10.5 g (7M)
Thiourea (F.W. 76.12)--- --- 3.8 g (2M)
CHAPS --- --- --- --- 1 g (4%)
DTT (F.W. 76.12)* --- --- 232.5 mg (60 mM) OR -
DeStreak Reagent (Amersham # 17-6003-18)*- 300 ?l
100x Bio-Lyte 3/10 ampholyte^ --- 125 ?l (0.5%) (Bio-Rad Cat. # 163-2094)
0.5% Bromophenol Blue --- --- --- 100 ?l (0.002 %)
H2O --- --- --- --- --- --- --- To make 25 ml final
Prepare and store as 0.5 ml aliquots at 20oC (without DTT/DeStreak Reagent and ampholytes).
*Just before use add the following:
(a) DTT 9.3 mg/ml OR - DeStreak Reagent as 12 ?l/ml.
(b) Ampholytes: 5 ?l/ml.

2. EQUILIBRATION BUFFER

Urea (F.W. 60.06) --- 36 g (6M)
SDS --- --- --- 2 g (2%)
1.5 M Tris-HCl (pH 8.8) 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

3. OVERLAY AGAROSE

Low-melt agarose 0.5 g (0.5%)
1x Tris-Glycine-SDS 100 ml
1% Bromophenol Blue 100 ?l of 1% (w/v) stock solution (.001%)

4. ELECTRODE BUFFER

Glycine --- 57.7 g
Tris --- 12.1 g
10% SDS --- 45 ml
Water to make 4 liters

5. STAINING SOLUTION
GelCode Blue Stain Reagent (Pierce Cat. # 24590) - or-
Simply Blue Safe Stain (Invitrogen Cat. #46-5034)

6. GEL DRYING SOLUTION
Glycerol --- 100 ml (10%)
Ethanol --- 200 ml (20%)
Water --- 700 ml



Supplies
Urea, Thiourea, CHAPS, DTT, SDS, Glycerol, Tris, Glycine, Methanol, Ethanol, Agarose, Bromophenol blue, Acetic Acid, Sodium Thiosulfate, Silver Nitrate, Formaldehyde, Sodium Chloride, Sodium Carbonate, Formalin, can be purchased from Sigma Chemical Company or any other general laboratory chemical suppliers. DeStreak reagent, 2D-Clean up kit, mineral oil was purchased from Amersham Biosciences. IPG (Immobilized pH Gradient) strips, ampholytes, Electrode Wicks, pre-cast second dimension SDS electrophoresis gels, were purchased from Bio-Rad Laboratories. GelCode Blue and Simply Blue (both Coomassie based) stains were purchased from Pierce Chemical Company and Invitrogen respectively.

Tips
(a) Use only "THICK" mineral oil (US Biochemicals Cat.# US71600. This can also be purchased directly from Amersham Pharmacia. Amersham Pharmacia Cat. # US71600-1l). "THIN" mineral oil easily moves from well to well and may result in drying/burning of IPG strips during IEF.

(b) Ampholytes exceeding 0.5% may cause IPG strip to burn during IEF due to excess salt concentration.

(c) IPG strips are available with different pH range (e.g., 3-10, 5-8, 4-7, 7-10, etc.) and different sizes (e.g., 7, 11, 17, 18, 24 cm, etc.). Similarly pre-cast linear or gradeint second dimensional polyacrylamide gels can be purchased in different sizes to accommodate a particular size of IPG strips.

Submitted by: gheda