Western BlottingThis 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 @ 4oC) 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 @ 4oC 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.