Team: University of Chicago/Notebook/SDS-PAGE

SDS-PAGE Protocol

Materials

polyacrylamide gel

1X SDS buffer (6.05g Tris, 28.83g glycine, 2g SDS per liter)

Benchmark Protein Ladder

Denatured protein samples

Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)

Destain solution (10% methanol, 10% acetic acid)

Procedures

1. Take the comb out of the prepared polyacrylamide gel. Place the gel in a holder and attach the holder to the PAGE apparatus.

2. Insert the apparatus in the tank and pour 1X SDS buffer in the tank and inside the apparatus. 3. Pipette 10ul of Benchmark ladder in one lane of the gel

4. Pipette desired amounts of protein samples in other lanes of the gel.

5. Put the lid on and connect the cables to the power supply

6. Set the power supply to 80V. Run for 20 minutes or until the dye front passes the stack.

7. Increase the voltage to 120V and continue running until the dye front reaches the end of the gel.

8. Cut off the power supply and remove gel from the apparatus.

9. Stain gel in commasie blue stain for 1h

10. Destain in destain solution for 30min.

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