genecutl at mendel.berkeley.edu
Mon Aug 1 15:16:06 EST 1994
In article <31ja1d$2vu at usenet.INS.CWRU.Edu>, pxb26 at po.CWRU.Edu (Peter
> 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.
Are you adding the competitor after the probe and protein? An obvious
thing is to mix the probe and competitor first and then add protien.
Otherwise, is it possible that your measurement of the concentration
of either the probe or the competitor?
Can you see any comptetion with nonspecific competitor? If you go up to
10,000 or 100,000 fold non-specific competitor you should be able to see
something happen. If the same effect is seen with your specific competitor,
then one of your DNAs is not what you think it is.
All of old. Nothing else ever. Ever tried. Ever failed.
No matter. Try again. Fail again. Fail better.
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