Run SDS gel.

Wear gloves. Cut 4 pieces of 3MM papers and a piece of nitrocellulose membrane at 7.5 x 8.5 cm for small gel.
Prepare 1 liter transfer buffer: 20% methanol,3.1 g/l Trizma base, 14.4 g/l Glycine. Pre-cool in cold room.
Under transfer buffer, put black side of cassette with one sponge then one 3MM paper. Transfer gel to one 3MM paper and place on top. Then membrane on top of gel followed by 2 pieces of 3MM. Remove bubbles by rolling a 15ml conical over the "sandwich".
Orient cassette black to black in transfer box with bfr.
Run at 80V, 1-2 hours or 25V o/n at 4C with stir bar on low.
(Optional: Check membrane with Ponceau Red. Wash with Blot buffer)
Transfer membrane to Blot bfr/10% milk 45 min-o/n(4C)
Incubate o/n(4C) with primary Ab in Blot bfr/5% milk. Reuse frozen stocks containing 0.02% NaAz.
Wash 2x5 min with Blot bfr.
Incubate at RT 1-4 h with secondary Ab in Blot bfr/5%milk.
Wash 3x10 min with blot bfr.

In dark room:
Incubate with 10ml total chemiluminescence reagent 1 min.
Wick excess on paper towel, but don't dry out!!
Wrap in Saram Wrap or put between plastic sheets. Use autorad tape (or other means to orient film)
Expose to XAR film 1 min and evaluate for longer or shorter exposure. (Signal has a half-life of 5-10 mins.)

Stripping:
Incubate in water 15 min.
Incubate in 0.1M NaOH, 15 min.
Incubate in water 15 min.
Block in Blot buffer/10% milk, 30min+

Blot buffer:
100 mM Tris HCl pH 7.5
150 mM NaCl
0.1% Tween 20