This is my first post here. I'm having trouble with EMSA and I was hoping you could help me. I'm doing EMSA with 10ug of nuclear extract got using the Thermoscientific Pierce kit, and using 0.5uM of a 150-260bp PCR amplified and gel purified or column purified 5'6-FAM labeled product as my probe. The probes have been eluted in Tris-HCl, EDTA and Nacl, and HEPES or Tris based binding buffer used for the reactions. After 30min incubation at RT, I load half or more of the reaction volume onto Novex non-denaturing 6% gels and run the gel in 0.5X or 0.25X TBE for 1.5-2hours at RT. Then I use a gel analyzer to get an image of the gel.
I see that 80-90% of the probe is bound with the addition of nuclear extract, but the bands are at the very top of the gel wells, almost as if the protein-DNA mix is not entering the gel at all. This binding seems to be specific as addition of 25X-50X of the cold probe competes away all of the band at the top, and a non-specific competitor does not. Can anyone please tell me if this is normal, and what I can do to improve it? Am I using too much protein, which is causing protein-DNA aggregates or is it possible my PCR fragments are too big. Irrespective of what conditions I change, this pattern remains unchanged with small differences in intensity of the bands at the top. I would greatly appreciate it if somebody can help me with this. Thanks!!