Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

Do cell signaling antibodies work with 1h RT incubation? - (Apr/06/2013 )

I am doing a western for cleaved caspase 3, and I always do a overnight 4oC incubation with cell signaling primary abs. Does anybody know if I can get same results with 1 or 2h at room temp? I need to finish seriesof expts within a week, and each depends on the previous expt, so would like to save time. Thanks for your help!


i didnt tried cleaved caspase3, but i am sure most antibodies frm cell signalling works fine in RT for 3hrs incubation, i usually decide based on the the signal intensity, for instance, ERK total ll give strong signal on overnight incub, so i use to put them at RT for 1hr.


It will depend on how much of your protein is in the sample and the antibody but the short answer is yes it can work. I've probed for lots of different proteins (including some other cell signaling one) using 1 hr incubation with the primary and 1 hour with the secondary at room temp. It usually works, otherwise overnight can give a stronger signal.