I am working on transcription factor characterization. I am using biotynalated, PCR-amplified promoter region and His-Tagged protein which is purified by using affinity chromatography. I have clearly observed the DNA: protein interaction by EMSA and that part is okay.
When I used chemicals /effectors/ligands to check the effect , one of the ligands showed strange result I got a shift band and no unbound DNA (as seen from the control) at highest ligand concentration (1mM) whereas at 0.5mM,0.25mM and 0.01mM there was binding and I could see both shift and unbound DNA.
Next time when I used the same protein (now a day old stored at 4 deg) and carried out EMSA with the 1mM ligand there was no shift but a control whereas at other concentrations result was consistent.
When I used new protein aliquot from -80 deg I got same shift but no unbound DNA at 1mM.
Can anybody tell the possible reason?
Is it the biotynalated promoter part getting masked after DNA: protein: effector interaction so that there is no super shift signal but just a shift because of DNA: protein interaction since this is the just DNA: protein and no effector is available since it is already complexed but the DNA is not visible since its biotynalated part is masked because of the interaction?