Western Blot Protocol using Novex Gels: 11/99

Prepare 800 mL of Running Buffer: 40 mL of 20X running buffer up to 800 mL.

Add 600 mL of running buffer to lower (outer) chamber.

Add 500 uL (0.25%) of antioxidant to 200 mL of running buffer for upper (inner) chamber only if use reducing agent below (recommended).

Add 1/10th volume of reducing agent to each sample (not markers).

Add 1/4th volume of loading buffer (4X) to each sample (not markers).

Heat samples at 70'C for 10 minutes.

Spin down the load samples (<25ul each).

Run for 50 minutes at 200V.

If running reduced samples, must add 10% (per gel) of methanol and 0.1% antioxidant to the Transfer buffer: 40 mL of 20X transfer buffer, 80 mL Methanol, 800ul antioxidant up to 800 mL.

Transfer at 30V for 1 hour.