Pull-Down Assay, EMSA Assay: recombinant protein vs nuclear extracts - (Apr/07/2015 )
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.
Why??
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?
Thank you
AntonioSillo on Tue Apr 7 11:55:07 2015 said:
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?
do you also use reducing agent? if not then disulfide bonds are probably responsible for producing the dimer.
if you have then you may be boiling too long. even in the presence of sds, overboiling can cause aggregation. you can heat at 60-70C for 10-20 minutes instead of boiling.
thank you for replying me, yes, I've used DTT + SDS or Glycine-HCl pH 2 in my elution buffer.
when you use pH 2 glycine, do you neutralize immediately after collection? the protein may not be stable at low pH.
yes, i neutralize with TRIS
do you then dialyze or otherwise reduce the ionic strength of the solution?
high ionic strength can cause all sorts of complications with page.