More question about gel-shift assay

[Andre Nantel]

In article <3DIJ99$xL1 at bbs.life.nthu.edu.tw>
klebs.bbs at bbs.life.nthu.edu.tw (Leo) writes:

>  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.

I tend to agree with the other responses, 500 bp is pretty big. You
could also try to switch to a Tris-Glycine buffer (25mM Tris, 190mM
glycine, 1mM EDTA, pH 8.3) and to increase your
acrylamide:bisacrylamide ration to 40:1. In addition, adding 2.5%
glycerol has helped in some cases.

Do you add bromophenol blue to your buffer? Many people do although
we've seen it inhibit the binding of several proteins. 

Good luck...and tell us how it works.

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Andre Nantel                             Purveyor of fine genetically-
Biotech Research Institute               engineered roadkills for the
National Research Council Canada         Information Superhighway
Montreal, Quebec
nantel at biotech.lan.nrc.ca or andre at bri.nrc.ca
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