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

newsnet customer via methods%40net.bio.net (by customer from newsnet.com)
Mon Mar 19 03:44:22 EST 2007


>>Dear experts,
>>
>>When I incubate two or more membranes with an antibody solution (eg O/
>>N at 4degC with gentle agitation) and the membranes stick together, in
>>worst case face to face, will the antibodies still reach their target
>>proteins and stick there?
>>
>>In my imagination, the membrane actually has big holes (0.2
>>micrometers, that's the same one can use for sterile filtration of
>>antibody solution) that should allow the antibodies to go everywhere,
>>if there is just enough time for diffusion.
>>
>>Right or wrong? How much time should one allow to be on the safe side?

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.

POINT 2:
Why would you incubate membranes face to face?
Incubate each membrane separately unless you do not have enough solution.





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