Western Blotting

Fill a tray with blotting buffer, remove the gel from the cassette using the opening key, trim the top and bottom of the gel, and equilibrate the gel in the buffer for 15 minutes on a rocking platform.

Pre-soak fiber pads in blotting buffer. Place the gel holder in a container with buffer, then layer one fiber pad, blotting paper, the gel, the nitrocellulose membrane, another blotting paper, and a second fiber pad, removing air bubbles between each layer.

Fold the clear plastic side of the gel holder and clamp it with the white clip, insert the gel holder into the inner module with black sides aligned, place the module in the electrophoresis chamber with a frozen cooling unit, and fill with blotting buffer.

Place the lid on the chamber, connect the electrical leads to the power supply (red to red, black to black), and run the blot at 20 volts for approximately 2.5 hours.

Turn off the power supply, disassemble the chamber, and remove the inner module. Open the module in a buffer-filled container and carefully remove each layer until the nitrocellulose membrane is exposed.

Immerse the membrane in 25 mL of blocking solution for 15 minutes on a rocking platform. Add 10 mL of primary antibody and incubate for 10-20 minutes, then perform a quick rinse and a 3-minute wash in wash buffer.

Add 10 mL of secondary antibody and incubate for 5-15 minutes on the rocking platform at fast speed. Perform a quick rinse in wash buffer, followed by a 3-minute wash in fresh wash buffer.

Add 10 mL of substrate and incubate the membrane for 10-30 minutes while watching for color development. Rinse twice with distilled water, blot dry with paper towel, air dry for 3-60 minutes, and cover in plastic wrap for storage.