I have problems with EMSA with DIG labeled oligos.... when I do electroblotting at 4 mA/cm2 then the volts and watts quickly increase, I've tried 0.5 X TBE and 1xTris-Glycine. And then blotting is not quantitative... when I use 2 mA/cm2 is OK, but the signal is very low and I don't see any shifts. It is strange because the signal of labeled oligo + 100x unlabeled oligo is much stronger than only labeled oligo.... Is it possible that the small fragments go through membrane when I blott too long (they are 29 bp long).
non isotopic EMSA
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