[Western Blotting] Diffusion of Antibodies in PVDF and NZ membranes

[Kyle Legate]

newsnet customer wrote:
> 
> POINT 1:
> Incubating the membrane in an antibody solution overnight seems to be overly 
> long.
> The antibody should bind to the protein, but the protein on the membrane may 
> diffuse into the solution.
> Therefore you will get reduced signal on your membrane.
> I think 2-3 hours is enough.
> 
Are you a troll? You do know that in this newsgroup it's better to give 
no information than to give false information, right?

Incubating membranes in antibody overnight is routine, and is in fact 
*required* for many antibodies--EGFR antibodies from Cell Signaling, for 
example. Also, once proteins are bound to nitrocellulose or PVDF 
membranes the interaction is essentially irreversible under normal 
antibody incubation conditions. Stripping membranes for reprobing 
involves much harsher conditions and proteins still remain bound to the 
membrane.

Having said that, incubating two blots in the same vessel is in general 
a Bad Idea; avoid it if possible, since regions of overlap will either 
have no signal or will be all signal.