Primary antibody - proteins (Nov/22/2004 )

well i want to know the minimum time requried to incubate the membrane with primary antibody while doing western and the stringency of the the washes i mean washing time and washing buffer volume,

can anybody tell me about that?

thanks

well, the minimum time for primary can be as low as 20min, but i do recommend 60min. The washes, I do 3 times 20min in a 30-50ml for a minigel, but you can do shorter times, depends on your antibody. Nice western is a balance between high signal and low background, so if you get no background at all, you can easily shorten your incubation times, whereas higher background, you can either do longer washes, higher salt/tween/milk, or you can lower your concentration of primary...

have fun,