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

[newsnet customer]

>> 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.

Factors that will influence protein binding affinity to membrane:
1. type of membrane.
2. transfer method
3. type of protein.

I have experienced protein diffusion from my membrane after an overnight 
antibody incubation. This was confirmed with a positive control. And since 
he had no signal or reduced signal on his membrane, this could be a possible 
explanation. Please realise that comments on a newsgroup should be taken 
with a grain of salt. And you should take a chill pill.

ST