EMSA - Protein-DNA complex is stuck in the well (May/16/2007 )
Since past few months i am trying to study the interaction of my protein of interest with DNA. The purified recombinant protent is more than 100 kda in size and has a PI of 8.6.When i run the 4% native gel for the EMSA all the protein-DNA complex stucks in the well. I have used different concentations of the protein starting from 5ng to 200 ng. In all the cases the complex stucks in the well. When i run the protein alone in the same gel in 1x TBE the protein does not enter the gel as its PI is equal to the PH of the running buffer. But when i run the protein in the native gel using the rumnning buffer PH 6.1, the protein of course enters the gel , but this buffer does not help me in the EMSA as DNA probe never enters the gel with this running bufer of PH 6.1 as DNA has extensively negative charge.
It is important to note that Protein is active in other assays.
Is anybody there to suggest to overcome this problem.
you could titrate some BSA into your binding buffer, and also into your running buffer if necessary. this can help the protein-DNA complex enter the well properly