Protein Gel Electrophoresis

PROTEIN GEL ELECTROPHORESIS

Preparation of the pre-cast gels:

1. Remove the Ready Gel from the storage pouch.

2. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer.

3.Cut along the dotted line at the bottom of the Ready Gel Cassette with a razor blade.

4. Pull the clear tape at the bottom of the Ready Gel Cassette to expose the bottom edge of the gel.

5. Repeat for second Ready Gel.

Mini-PROTEAN 3 Electrophoresis Module Assembly and Sample Loading (see figure 5):

1. Place a Gel Cassette Sandwich into the slots at the bottom of each side of the Electrode Assembly. Be sure the Short Plate of the Gel Cassette Sandwich faces inward toward the notches of the U-shaped gaskets (see Figure 5b).

2. Lift the Gel Cassette Sandwich into place against the green gaskets and slide into the Clamping Frame (see Figure 5c).

3. Press down on the Electrode Assembly while closing the two cam levers of the Camping Frame to form the Inner Chamber and to insure a proper seal of the short plate against the notch on the U-shaped gasket. (see Figure 5d). Short plate must align with notch in gasket.

4. Lower the Inner Chamber Assembly. Fill the inner chamber with ~ 125 ml of 1X running buffer (until the level reaches halfway between the tops of the taller and the shorter glass plates of the gel cassettes). DO NOT OVERFILL THE INNER CHAMBER ASSEMBLY!! Add ~ 200 ml of 1X running buffer to the Mini Tank (lower buffer chamber).

5. Load 30µl of standard or sample/well (35 µg total protein/well). NOTE: Bring all samples to 30µl.

6. Place lid on mini-tank

7. Attach leads to power supply.

8. Run at 160 volts, constant voltage, for 45 minutes.

Gel Removal:

1. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.

2. Remove the tank lid and carefully lift out the Inner Chamber Assembly. Pour off and

discard the running buffer.

Note: Always pour off the buffer before opening the cams to avoid spilling the buffer.

3. Open the cams of the Clamping Frame. Pull the Electrode Assembly out of the Clamping

Frame and remove the Gel Cassette Sandwiches.

4. Remove the gels from the Gel Cassette Sandwich by gently separating the two plates of the gel cassette. The green, wedge shaped, plastic Gel Releaser may be used to help pry the glass plates apart.

Note: To remove the gel from a Ready Gel Cassette, first slice the tape along the sides

of the Ready Gel Cassette where the inner glass plate meets the outer plastic plate.

5. Run the sharp edge of the Gel Releaser or a razor blade along each spacer to separate the gel from the spacer. Remove the gel by floating it off the glass plate by inverting the gel and plate under fixative or transfer solution, agitating gently until the gel separates from the plate.

6. Rinse the Mini-PROTEAN 3 cell electrode assembly, Clamping Frame and Mini Tank

with distilled, deionized water after use.