Hi. Couple weeks ago I started EMSA. My RNA is 260nt long and the protein molecular weight is 65 kDa. I attached the gel and I need Your help with interpretation. First I separate my samples on 8% gel (40:1) - left panel and recently I separated samples on 6% gel (75:1)- right panel. As You can see I obtained different results. I marked free RNA and probably the RNA-protein complex but can anyone explain me what is the band marked with ,,?''. I dont think so that is a complex with protein because is to close to free RNA and the protein is quite big. So what is it then?
The gel is a native PAA (50mM Tris/ 50mM glycine at ph 8.3)
Is it possible that band marked with ,,?'' comigrate with the RNA-protein complex on 8% gel because of the 40:1 ratio of acrylamide/bisacrylamide?
Thanks in advance