I want to determined the Kd of the binding of ~400nt long RNA and a peptide which has 13 aminoacid residues.
I did a filter binding assay which shows a binding and Kd ~17uM.
As a assume that the peptide can bind in multiple sites, I want to see this in EMSA. I did 4% PAGE (acryl:bisacryl 80:1) native with 0.5mM DTT and 5% glicerol, 0.5x TBE and run a gel in 4C but I don't see any shift using the same range of peptide concentration as in filter binding assay.
My peptide has a Iso-electric point: pH 8.5
The binding conditions are: 10mM Tris-Hcl, pH 7.4; 25mM KCl; 0.5mM DTT; 50ug/ml BSA; RNase Inhibitor ; incubation 30min/ 4C. I assume that since I see the binding on the filter, the conditions of binding are OK, and the trouble is with electrophoresis.
Could it be that the peptide is so small, that I would not see a shift with a 400nt long probe? I can not shorten it.
Any advice on the electrophoretic conditions? Would agarose be better that acryalmide gel?
I would be grateful for any advice.