gel-shift problem

xiaokezh at kuhub.cc.ukans.edu xiaokezh at kuhub.cc.ukans.edu
Tue Oct 3 22:04:47 EST 1995


Dear collegues:
I have encountered a problem with my gel-shift experiments and urgently need
your kind help. I have been trying to do a gel-shift using a 31 base pair oligo
which is likely to be a GAS (gamma-IFN activated sequence). When I used 10 ug
of whole cell extract to do the gel-shift, I couldn't detect any bands.
However, by adding anti-Stat1 alpha antibody (dimers of phosphorylated Stat1
alpha are known to bind to GAS), I could get a clear super-shift. In order to
increase binding affinity, I also used an oligo that contained 4 copies of the
GAS and a nuclear extract to do the gel shift. I got a much stronger
super-shift band using anti-Stat1 alpha antibody, but was still unable to
detect a gel-shift band. I cannot figure out the reason for or solution to this
problem. If anyone could give some suggestions, I would highly appreciate it. I
await your reply. My email address is: gaozuema at kuhub.cc.ukans.edu 
zdetect 



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