EMSA Supershift question - Bigger signal but no supershift (Aug/14/2007 )

Hi all, I have a question concerning supershift in gelshift-assay (EMSA). When I try to get a super-shift and add my antibody to the nuclear extract in binding buffer, incubate at RT for 30 min, then add the probe and incubate once more for 30 min at RT, I only see the same shift-signal which a get without antibody. I only gets fatter the more antibody I add to the mix. It's the same thing when i reverse the procedure, first probe, than antibody. Also, signal looks good but won't shift higher, just gets bigger....

Anybody get any ideas???

You're right, it is odd. I always used to do my EMSAs on ice for the binding, and in the cold room for the gel. Perhaps your complex isn't stable?
Alternately, you could change your buffers. Some complexes are sensitive to the amount of phosphate, salt etc, even the pH.

I'm with Swanny. monkey around with your buffer composition. BSA can be very important...also, try different incubation times/temps.

if all else fails - check your antibody. has it been used in EMSA before? is it binding to an epitope that interferes with the DNA-TF binding, in such a way that you'll never see a supershift? perhaps you need to try an antibody recognizing a different epitope on the TF.

good luck