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NF-kB EMSA - Different nuclear protein extraction method leads to opposite results (Sep/13/2006 )


I tried two kinds of method in nuclear prep.
One is following Molecular Cloning, using Dounce Homogenizer and the final concentration of NP-40 in cytoplasmic extraction buffer is 0.05%. The NF-kB activity is upregulated following drug treatment using this nuclear prep.
In the other method, I didn't use Dounce Homogenizer and I added NP-40 to a final concentration of 0.3% after swelling cells. Using this nuclear prep, the NF-kB activity is downregulated following drug treatment.
I am trying to figure out what's the problem.
Thank you for your help!


have you done any sort of control to determine how much/little cytoplasmic protein contamination you may have?

if you have very much cytoplasmic protein in one of your preps, perhaps the NFKB that hasn't yet undergone nuclear translocation could be giving you false positives?