EMSA- high background and smear shift - seeking help (Jan/16/2006 )
There is a trouble about my EMSA. I'm using 0.2 pmol double-strand-p32-labeled oligo to incubate with 6.6 ug cell nuclear extract and then using 5% polyacrylamide gel (pH 7.5) to perform the EMSA. Eventhough I have included 4ug poly dI.dC in the binding mix, the upshifting band still appear as a smear, and the lanes have a high background.
I have tried to reduce the percentage of glycerol to 2% in order to lower the smearing effect, but it does not improved.
Could anybody give me some suggestions? Thank you.
what about degradation of your protein (transcription factor)? do you include appropriate protease inhibitors in your nuclear extraction protocol?
another thing, are you certain of your oligo specificity? also, are you certain that only one transcription factor complex binds at your sequence of interest? many TF families are comprised of many proteins in varying combinations that can sometimes bind the same sequence motifs with differing specificity...
How long is your oligonucleotide probe. In my experience, the longer the probe the more smearing and background you get. I've tended to use oligos between 20-50 bps (most protein binding sites will be easily accommodated in these).
Also, you can try different acrylamide:bis-acrylamide ratios to reduce the amount of crosslinking in the gel matrix. Most people tend to just get the 19:1 mix but I've found that a 37.5:1 mix helps band migrate more smoothly.
For some proteins that appear problematic at first, 0.1% Triton X-100 (final) seems to help.
There are plenty of things to look at. What about the apparatus? The glass plates you're using, the electrophoresis conditions, etc.
Finally, what running buffer are you using? Does this match identically with what's in your acrylamide gel?