More question about gel-shift assay
klebs.bbs at bbs.life.nthu.edu.tw
Sat Jun 22 10:09:44 EST 1996
I am working on two different bacterial transcription factors.
One is very easy to handle. I can get beautiful gel-shift
pictures easily. However, I just can't get any discrete
band out of the second transcription factor. It always yielded
a smear pattern. When you increase the protein concentration,
the DNA/protein complex just stuck in the wells. The protein
is purified through His-Bind column and has been dialyzed.
The probe is approximately 500 bp. Our typical buffer is
50 mM HEPES pH7.8, 100 mM KCl and 2 mM MgCl2. We run the gel
in 1 x TBE buffer at 4 degree C. The binding
, although smear, seems specific. Adding 50 x quantity of pBR322
DNA in the reaction did not affect the smear pattern.
I will be very grateful if any of you can provide me some thoughts
to improve my experiment. Thank you.
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