EMSA-Competition problem

Peter Brunovskis pxb26 at po.CWRU.Edu
Mon Aug 1 12:05:49 EST 1994


HELP!!!
I am having a problem that is driving me nuts!

I am working with a Ni-His-purified bZIP factor using 
commercially obtained AP1 oligonucleotide probes labeled with 
T4 PNK. The problem is I cannot compete away DNA-protein
complexes with 100-1000-fold excess of the same unlabeled oligo,
even under conditions in which protein is NOT in excess of 
labeled probe.  I have tried different salt concentrations,
different oligo probes that bind this factor and different
polynucleotide carrier/non-specific competitors.  However, the 
same result is observed--no decrease in binding of labeled probe
to my factor.  The same result is observed with control bZIP
factors (purified the same way) with well-characterized binding
specificities.  These latter factors clearly form heterodimers
with my factor which exhibit cooperative binding to my labeled
probes, but they ALSO FAIL to be competed with excess unlabeled
oligo probe.
Can anyone tell me what I may be doing wrong or what I may try
to alleviate this problem?
I greatly appreciate any and all responses.



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