Hi to all,
It's The first time in this fantastic forum!
I think that you can help me.
I'd like to study by NMR a protein-DNA complex.
It's well known, as reported in several papers, that a protein found in mouse embryonic fibroblast interact with a portion of 30 bp of DNA.
So I've repeated with EMSA Assay this experiment using Human Embryonic Nuclear Protein extracts.
I had positive results. I repeated this experiment with the recombinant protein known to interact with the 30 bp DNA probe, but I had no positive results. I tried to study this interaction also by NMR, but still no trace of any interaction.
I tried to digest the pulled down protein within the nuclear extracts, in order to look for any post translational modifications..., but the nuclear protein seems not to undergo to any PTM.
I tried another assay: I used Streptavidin coated magnetic beads, and I observe a positive result both with recombinant protein and with nuclear protein.
Furthermore the protein, eluted with various methods (SDS Boiling, Tris glycine.....) Seems to band at a MW compatible with a dimer?
How is it possible since I've used denaturing conditions?