EMSA/band shift assay with RNA probes

Jochen jochen at iigb.na.cnr.it
Tue Jul 24 11:36:26 EST 2001


I'm trying to get a band shift with cytoplasmic and nuclear extracts 
from HeLa cells using RNA probes for binding. Actually I'm getting a 
band shift, but the problem is that there are two bands, same for 
each kind of extract, but they are appearing and disappearing in an 
irregular manner, meaning one time i have two bands in the nuclear 
assay and another time just one. for the cytoplasmic extracts it's 
even more confusing because i get either band or nothing.
The upper band has the height of the undigested probe, but the the 
digestion control for the rnase was ok, so i didn't suppose it might 
be the undigested probe, but now i get some doubts on that. 
Eventually that would mean that the upper band is an artefact, and 
actually i wasn't able to do a competition on it yet, but for the 
lower band there was a competition.
So i would be very pleased if someone could tell me if the band with 
height of the probe might be the undigested probe and if different 
concentrations of protein and/or probe might change the resulting 
bands.

Thanks a lot in advance,
Jochen

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