Supershift/EMSA problem - (Jul/20/2005 )

Hi

I'm having trouble with my EMSAs.

I've been adding the target antibody to cell nuclear extracts and incubating, followed by the addition of the 32P labelled oligo, hoping to see immunodepletion of the protein compared to samples with cell nuclear extracts plus oligo alone. However, the antibody is not binding to the protein in my positive control samples. So when I run it on a gel the nuc extract + oligo is the same intensity as nuc extract + antibody + oligo. I have tried incubating at 4 deg overnight and also at room temperature (which is quite high at the moment) for 45 mins and neither works. This system has worked before. Any suggestions?

The other thing that I am curious about is, when I add the oligo to the nuclear extract followed by addition of the antibody, there is no supershift but there is a much more intense band, the same size as the oligo + nuc extract and as the oligo + antibody + nuc extract. Any suggestions on whats happening?

Hope this isnt too confusing

Emma

Hi Emma,
My experience with EMSAs (for NFkB and STATs) is that the antibody determines whether you get a shift or a loss of binding, which I guess this depends on the epitope. We've always done pre-incubation with the antibody at room temperature for 20mins before adding the probe. Others in the lab have done similar things with AP-1 and SP-1.

I think the antibody is the critical factor. We generally use Santa Cruz antibodies which you can either use at the concentration provided for Western or a more concentrated version specifically made for supershifting. It's sometimes difficult to find out which exact antibody papers use as Santa Cruz make difficult clones.

What are you trying to shift?

Hope that helps,
Ceri