I have a quick question. How long do you all typically incubate your primary antibodies on PVDF transferred proteins for western blot purposes? The reason I ask is that I was accustomed to doing an overnight incubation at 4C in a roller but the new lab I work for actually does theirs at room temp for only an hour. Higher antibody concentrations than what I'm used (1:500) to but it seems to work for them. Is there any reason why overnight would be preferable to just 1 hour incubation length and is there any particular reason that most protocols call for a 4C incubation rather than room temp?
Also I know this second question depends a lot on the target quantity but how much protein do you folks usually load onto your gels?
Edited by cancergeek, 12 July 2009 - 02:53 PM.