reprobing a membrane to detect a protein with the same molecular weight (View forum version)



nocas

Posted 04 May 2010 - 08:11 AM

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

bob1

Posted 04 May 2010 - 04:50 PM

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...

zienpiggie

Posted 04 May 2010 - 09:00 PM

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."

olga_m

Posted 05 May 2010 - 01:15 AM

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

nocas

Posted 05 May 2010 - 01:17 AM

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

Thanks