I've run a number of RNA-protein and RNA-peptide EMSAs but this is my first sub-shift (complex running below free) result. I'm wondering if others have seen before? Google and literature have been little help and my labmates/PI are a bit shocked also. Is this suggesting a major structure change upon binding? (this would be cool) Should I be more worried about aggregation?

I've ruled out RNAse contamination after running denaturing gel of RNA-Protein complex, also have 3 controls of different binding and non-binding RNAs of same size on same gel with same protein sample added. The RNA showing the sub-shift is a 34mer stem loop with 2x1 internal loop and 18nt single strand region. The protein is 80AA with single RNA binding site.

Maybe run DLS to confirm not aggregation related problem? Maybe fluorecence anisotropy? Or DOSY-NMR?

I've tried different gel %'s (from 8-15%), different buffering systems, different bis/acrylamide ratios, adding detergents, tRNAs, different running temps, and voltages, gel thickness. All have helped sharpen the bands but the subshift is still there.

Thanks,

matt