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

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Mon Mar 19 17:21:57 EST 2007


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.

It is, if you do the incubation at 37 degrees (even 1 h is usually
enough). If you do the incubation at 4 degrees, incubation o/n is
appropriate. Advantage of this method: The antibody solution (which
should of course contain an anti-fouling agent like timerosal) can be
re-used many, many times without reduction in signal strength. With
valuable primary antibodies that can be important. I have done an entire
3 year postdoc with a single antibody solution.



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