reprobing a membrane to detect a protein with the same molecular weight - (May/04/2010 )

Hi,

I would like to know if it is correct to reprobe a membrane, after stripping, to detect a protein with the same molecular weight than the one that have been probed before. Someone told me that this isn't a correct procedure if the secondary antibody is the same. So I would like to hear other opinions.

Thanks

If the secondaries are the same, how will you determine that the first primary is all gone, and that the signal you are getting now is a result of the second primary...

after stripping, aren't there steps to ensure that the primary and secondary are both gone prior to reprobing? I bought restore stripping solution from fisher, and there was an instruction for that.

'Test for complete removal of the HRP label (e.g., secondary antibody): Incubate the membrane with new
SuperSignal West Working Solution and expose to film. If no signal is detected using a 5-minute exposure, the HRP
conjugate has been successfully removed from the antigen or primary antibody.


Test for complete removal of the primary antibody: Incubate the membrane with the HRP-labeled secondary
antibody, followed by a wash in wash buffer. Incubate in new SuperSignal West Working Solution and expose to
film. If no signal is detected with a 5-minute exposure, the primary antibody has been successfully removed from
the antigen."

I use to do it for two proteins at 120KDa but with different secondary antibodies (anti-mouse and anti-rabbit, HRP conjugated). But I'm still not sure if I do the right thing. What do you thing? Should I repeat the electrophoresis instead of reprobing the same membrane?

Thank u very much,
Olga

That's a good idea. I'll try that.

Thanks