Unable to reverse the shift in EMSA by unlabeled competitor - EMSA (Aug/03/2006 )

Hello everybody,
I am experiencing a very strange problem in my gel shift assay. I am using pierce chemiluminiscent EMSA kit. I am successful in getting the shift with plant crude nuclear extract using about 200 bp target biotinylated at one strand. DNA get shifted at 2 places, that may be due to multiple binding sites as 200 bp is a long target. My real problem is the cold probe. I have tried using its concentration ranging from 4 pmole to 16 p mole when I use 20 fmol of labeled probe. Strange enough when I increase the concentration, "I observed increased intensity of shifted band", i.e even more than real shifted band. Also, I always see a smear in the lane from high mol wt. DNA to low mol. wt. DNA in the competitor lane. I get no smears in the lane where I add the labeled probe and protein extract only, very clear shifted bands are obtained. I have tried doing EMSA several times diluting lableled probe and varying concentrations of cold probe and protein. I also incubate the cold probe with protein first for 15 min and then add the lableled probe...still I am unable to reverse the shifted band.
Somebody, please help me. Your views and expert comments are appreciated and awaited.
Thanks,
uhsna.

I don't know if this is helpful...but my first guess would be that you are fishing for DNA binding proteins that will bind to any old sequence. 200 bp is HUGE for EMSA; you will have all sorts of DNA binding motifs and partial motifs present in that oligo that will be recognized by various TFs and such

This explains the mess you see when you add cold probe. I am surprised that you only get two bands with labelled probe, and that they are crisp...I would not have expected that??

I'm sorry I can't help more, but hopefully someone else here can explain the crisp bands?