2D Gel Electrophoresis Protocol

Ensure there is enough or make up a stock solution of:

Sample Rehydration Buffer:

7M Urea 4.20 g
2M Thiorea 1.52 g
4% CHAPS 400 mg

Make up to 10ml in milli-Q water, store in 1ml aliquots at -20ºC.

Re-equilibration Buffer:

Tris HCl (1.5M stock pH 8.8 w/HCI) 13.4ml 50mM

Urea 144.14g 6M
Glycerol (87% stock) 138mL 30%
SDS 8.0g 2%
Bromophenol blue Trace (add with pipette tip)

Make up to 400ml in ddH2O, store in 40ml aliquots at –20ºC.

10x Running Buffer:

Tris Base 60.57g

Glycine 288.27g
SDS 20.00g

Adjust volume to 2L. pH ~8.5. Be sure to dilute down to 1x running buffer before using to run your
gels.

1. Determine protein concentration.

a. This can be estimated using an A280 reading or a quantification kit.

2. Sample preparation with Cleanup Kit on desired amount of protein.

a. In general gels need approximately 80-120ug of protein for silver staining.

3. Re-hydrate protein in rehydration buffer:

IPG Strip Length: Rehydration Buffer Needed:

7cm IPG Strip 125ul*

11cm IPG Strip 200ul*
18cm IPG Strip 350ul*
24cm IPG Strip 450ul*

*1mL Stock Rehydration Buffer needs the following added before use:
DTT 5 mg (=30mM)
Ampholytes 5ul (=0.5%)
Bromophenol blue Trace (add with pipette tip)

4. Pipette samples into rehydration tray, aiming for the middle of the lane. Try to avoid bubbles.

5. Remove protective cover from strip using tweezers.

6. Place strip gel side down into the rehydration well very carefully with the “+” or positive end of the
strip aligned with the “+” or positive end of the tray. Ensure that the length of the gel is covered in
sample. Raising and lowering the strip several times can help to spread the liquid, and all bubbles
under the strip should be avoided or removed.

7. Cover the strips with mineral oil to prevent them from drying out.

8. Rehydration and Focusing:

a. Rehydration should be active for 12 hours with 50v.

b. Focusing time depends on the length of the strip.

11cm IPG Strip 18cm IPG Strip

Suggested Program:
200v for 1 hour
1000v for 1 hour
4000v for 3 hour
8000v for 2 hour Suggested Program:
250v 1h
500v 1h
1000v 1h
4000v 3h
8000v 4h
Minimum Needed:
25000volt-hours Minimum Needed:
45000volt-hours

c. A removal window step can be added to the end of the focusing, we use 500v for 2 hours.

9. Remove IPG strip from rehydration tray, place gel side up on paper towel to remove oil from back
of strip.
10. Wipe the front of the IPG strip off very carefully dragging the edge of a Kimwipe along the length
of the strip, be careful not to press into the gel.

Note: Following focusing, strips can be frozen at –80 ºC if necessary due to lack of time. When ready
to continue: unfreeze and continue with the re-equilibration steps.

11. Re-equilibrate strips for second dimension in Bio-Rad re-equilibration tray which require
approximately 1.5mL-3mL per lane to cover a strip.

a. Prepare re-equilibration solutions.

Solution 1 10mL Stock Re-equilibration Buffer + 100mg DTT

Solution 2 10mL Stock Re-equilibration Buffer + 250mg iodacetamide

b. Cover strips with solution 1, gel side up, and soak strips on shaker for 20 minutes.

c. Decant solution 1.

d. Cover strips with solution 2, gel side up, and soak strips on shaker for 20 minutes.

e. Decant solution 2.

f. Cover strips with 1x running buffer, gel side up, and soak strips on shaker for 5 minutes.

12. Prepare agarose sealing solution:

a. 0.5% agarose in running buffer plus a trace of bromophenol blue.

b. Heat up in a microwave to make sure agarose is dissolved.

13. Ready 2nd Dimension tank:

a. Place pre-cast or in-house made gel into tank.

b. Remove combs from gel.

c. Fill tank with 1x running buffer up to fill line, ensure to rise wells with 1x running buffer.

14. Place strips into empty gel well and push down till it is flat against the bottom of the well and there
are no bubbles trapped underneath it. An old comb cut in half is a good tool to use to push strips into
place. Tweezers work just as well (be careful not to push too hard).
15. Pipette agarose sealing solution on top of strip to seal it into place.
16. Start running second dimension. Start the voltage off low ~50-80volts once bromophenol blue is
on to gel increase voltage to ~150volts. It is best to run until the bromophenol blue is off the end of
the gel.
17. Take gels out, disassemble cassettes.
18. Mark gels for identification purposes by trimming corners. Cut a large piece of at the top ”+” end.
19. Place gels into staining trays and stain according to silver protocol, make sure to get gels into fix
solution from silver staining protocol ASAP.