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protein did not enter the gel on EMSA - (Sep/26/2005 )

I am doing EMSA. but I found there is strong signal in the well. How does the protein:DNA complex not enter the gel, I use 5% non-denature polyacrylamide gel.


There can be several reasons, the most simple one is if your wells are not clean, do you wash it with a syringe? The second reason can be if your protein is forming oligomers or aggregates - is it a purified protein or extract? try lowering the concentration of the protein. Lastly use 4% gel to see if it helps.


I've got the same problem with mines. My EMSA are carried out with 120mM final NaCl. Perhaps, the ionic strength is not well gauged.
Nicolas From Paris