Set up the electrophoresis apparatus
- Retrieve one of the SDS-PAGE gels from the refrigerator.
- Carefully remove the comb from the spacer gel.
- Remove the casting frame from the gel cassette sandwich and place the sandwich against the gasket on one side of the electrode assembly, with the short plate facing inward. Place a second gel cassette or a buffer dam against the gasket in the other side of the electrode assembly.
- Clamp the green clamps on the sides of the electrode assembly (below).
- Lower the chamber into the electrophoresis tank.
- Fill the space between the two gels with Tris-glycine running buffer. This forms the upper chamber for electrophoresis.
- Add Tris-glycine running buffer to the outer (lower) chamber until the level is high enough to cover the platinum wire in the electrode assembly.
Load and run samples on the SDS-PAGE gel
- Retrieve your cell extracts from the freezer. Recall that the samples have already been mixed with a tracking dye and glycerol. Allow the extracts to thaw and vortex vigorously for ~ 10 seconds to thoroughly mix the contents.
- Using gel loading micropipette tips (tips have very long, thin points and fit P20s or P200s), load up to 15 μL of sample into each well. Load 5 μL of a molecular weight standard into one lane of the gel. Load samples slowly and allow the samples to settle evenly on the bottom of the well.
NOTE: Be sure to record the order of samples loaded onto the gel.
- Connect the tank to the power supply. Fit the tank cover onto the electrodes protruding up from the electrode assembly. Insert the electrical leads into the power supply outlets (connect black to black and red to red).
- Turn on the power supply. Run the gel at a constant voltage of 120‐150 V. Run the gel until the blue dye front nearly reaches the bottom of the gel. This may take between 45-60 min.