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question about EMSA - (May/04/2010 )

Hi all,

I am doing the gel shift assay (EMSA, protein-DNA interaction) for one of my proteins. I labeled my probe with biotin at 5'. I saw strong shift in protein + hot probe sample. However, when I tried to compete the binding with cold probe. I never see signal decreasing, instead I saw increasing binding. The binding reaction buffer I used is 15mM HEPES-KOH (pH7.5), 7.5mM KCl, 2mM DTT, 0.5mM EDTA, 6% Glycerol and 50ng/ul PolydI.dC. Anyone has the similar experience with me? Can you give me some explanation? Thank you so much! :lol:

-ninitutu-

ninitutu on May 4 2010, 02:40 PM said:

Hi all,

I am doing the gel shift assay (EMSA, protein-DNA interaction) for one of my proteins. I labeled my probe with biotin at 5'. I saw strong shift in protein + hot probe sample. However, when I tried to compete the binding with cold probe. I never see signal decreasing, instead I saw increasing binding. The binding reaction buffer I used is 15mM HEPES-KOH (pH7.5), 7.5mM KCl, 2mM DTT, 0.5mM EDTA, 6% Glycerol and 50ng/ul PolydI.dC. Anyone has the similar experience with me? Can you give me some explanation? Thank you so much! :P



Did you run a control without protein? I am using DIG to label, because my plant has endogenous bitotin. I got similar band to yours. but I found that is a artifact caused by the probe per se. By the way, are you using whole cell nuclear extract?

-huotoad-