Redoing only the Secondary Antibody staining on a Western - (Jan/28/2012 )

Hi all,

I was wondering what the thought was about redoing only the 2ndary antibody step on a Western Film.

During my first attempt at staining my gel, my film was too dark and I couldn't see individual bands very well.

The next time, I stripped the film, redid the primary step at the same concentration, and made my 2ndary 3X more dilute. When I developed the film, I didn't see anything. I'm guessing this was due to a low concentration of 2ndary antibody.

Since I didn't see anything from my film, I'm wondering if I could simply rinse off the membrane (to remove the developing solution, et. al) and simply incubate with a more secondary, this time at a higher concentration than my last round, but a lower concentration than my first.

Any input would be appreciated.

Yes, you can do that. In my opinion, stripping a blot is not a good option for these sorts of things, you would have been better off quenching the HRP (presuming you are using it, not alkaline phosphatase) using azide.