Hello colleaques,
I have a strange problem with my EMSAs I cannot explain, thus I would like to ask for an advice from more experienced in EMSA. I am performing my EMSAs using biotinylated oligos according to the instructions provided by PIERCE (LightShift Chemiluminescent EMSA Kit). For my experiments (focused on NFkB) I use both nuclear extracts and purified recombinant proteins (from Active Motif). In both cases I get no shifted bands but in majority of cases (including wild type NFkB recognition sequence) I can observe that in lane with shift reaction I have much less(~90%) of biotinylated DNA as in free probe (without protein) and in competition reaction (with protein + 200X excess of unlabeled DNA). This would mean that my DNA was bound by protein, why than I can not see shifted band?? I was thinking that in case of nuclear extracts my DNA could be bound by a different NFkB complexes what would finally "dilute" my biotinylated oligo so far that it would not be detected anymore.. The problem is that I have the same situation with purified recombinant proteins.. Has anybody an idea what's going on??
Maciek
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