why some antibodies need overnight incubation for Western blot - (Aug/22/2006 )

why is that some antibodies are recommended to be incubated for overnight for Wblot?
what difference does it make whether to incubate at RT for 2 hrs of overnight at 4C??? probably something related to affinity or something????
thanks.

Yup, affinity is the answer. Good WB result depends on many things, blocking reagent, Ab dilution, mono vs polyclonal Ab, Ab providing company etc. For good Ab 2 hr at RT is enough but should be used only when you are in hurry to get result. For most Ab overnight is a better option.

i read once that as a general role of incubation in WB is:
30 minutes in 37 degree or 1 hour in room temperature or overnight in 4 degree BUT
in the case of first antibody the higher temperature isnot recommended as it will increase the binding to protein both specific and nonspecfic noise protein so its for the best result to make the incubation for first antibody overnight in 4 degree.
hope this will help.

please read this

page 8

http://www.abcam.com/assets/pdf/protocols/WB-beginner.pdf

thanks a lot guys,
i appreciate ur comments.