More question about gel-shift assay

Leo 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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