Western Blotting

This Protocol starts where your SDS-PAGE or Native-Gel ends

Transfer Buffer 4L (Store in a cold room)

Trizma base 12.12g (0.303%)

Glycine 57.6g (1.44%)

SDS (optional) 2g (0.05%)

Methanol 800ml (20%)

TBST 4L (Store @RT)

1M Tris 7.4 50ml

NaCl 29.2g (0.0073%)

Tween20 2ml (0.05%)

ECL Mix 500ml (Store in dark @ 4^oC)

1M Tris 8.3 20ml

Luminal stock (11% in DMSO) 1ml

p-Coumaric Acid Stock (1.7% in DMSO) 1ml

Protocol

· Transfer the proteins @ 50volts (400mAmps) for 1 hours in cold room.

· After transfer briefly rinse the blot once in ddH₂O and once in TBST.

· Block in 5% Non Fat dry milk (NFDM) in TBST at least for an hour @RT on a shaker.

· Incubate in the primary antibody diluted in TBST with 2.5% NFDM @RT for 2 hours or @ 4^oC O/N on a shaker.

· Wash three times 5min each in TBST on a shaker.

· Incubate in the secondary antibody diluted in TBST with 2.5% NFDM @RT for 1hour on a shaker.

· Wash three times 10 min each in TBST on a shaker.

· Add 3 m l of Hydrogen Peroxide to 10ml of ECL mix and incubate the antibody probed blot for at least one minute.

· Wrap the blot in a Saran Wrap, squeeze out the excess reagent with a kim-wipe and expose it to film.

· Develop the film.

· BINGO!!!! J YOU GOT THOSE BEAUTIFUL BANDS.