run gels as required for separation of your favorite proteins
if you have prestained size markers, use them! otherwise use unstained markers and paint them after blotting

semi-dry blots

- sandwich 3 layers of 3M-paper, nitrocellulose- or PVDF membrane, gel, another 3 layers of 3M-paper
- paper and membrane should be the same size as the gel. if bigger, seal overhangs with parafilm or strips of film
- paper, membrane and gel should be thoroughly wetted with Towbin buffer. PVDF should be pre-wetted in methanol
- remove air bubbes by rolling a pipet over the stack
- blot at maximum volts, 0.8 mA per square cm of blot for 1 h. proteins run to the positive electrode!
- alternative numbers: blot 1 h at up to 400 mA or o/n at 20 mA

Towbin buffer: 25 mM Tris, 192 mM glycin, 20% methanol, pH 8.3
alternative buffer: 14.6 g glycin, 15 ml acetic acid, water to 1 liter

wet blots

- sandwich 3 layers of 3M-paper, nitrocellulose- or PVDF membrane, gel, another 3 layers of 3M-paper
- paper and membrane should be the same size as the gel. if bigger, seal overhangs with parafilm or strips of film
- paper, membrane and gel should be thoroughly wetted with wet blot buffer. PVDF should be pre-wetted in methanol
- remove air bubbes by rolling a pipet over the stack
- blot at maximum volts, 100 mA, o/n. proteins run to the positive electrode!

wet blot buffer:  14 g glycin, 3 g Tris, 200 ml methanol, water to 1 liter

staining proteins on blots

ponceau:

- stain 1 to 3 min in 0.2% Ponceau S in 4% trichloroacetic acid
- remove excess stain with PBS or TBS. stain from protein bands is washed out during blocking

amido black:

- stain 3 min in 0.1% amido black, 10% methanol, 2% aceticacid
- destain in 40 % methanol, 10% acetic acid

coomassie blue:

- stain 2 min in 0.1% coomassie blue, 50% methanol, 10% acetic acid
- destain in 50% methanol, 10% acetic acid

blocking and detection

- incubate 30 min to o/n in 5 % non-fat dry milk in TBS or PBS (alternatively, block in 1% BSA in TBS or PBS)
- incubate 30 min to 1 hour with 1st antibody in blocking solution (or incubate o/n at 4 C)
- wash 3x 15 min in TBS or PBS. if desired, add Tween to 0.1-0.3% (some primary antibodies require more/longer washes)
- incubate 30 min with 2nd antibody in blocking solution (or incubate o/n at 4 C)
- wash 3x 15 min in TBS or PBS. if desired, add Tween to 0.1-0.3%
- detect antibodies with ECL, CSPD or NBT/BCIP according to the manufacterers instructions