DNA/protein mobility shift assay

Warren watchcity at my-deja.com
Sat Jan 22 20:34:23 EST 2000


Hello, I have done a lot of RNA:Protein work, and some of the things you may
wish to test: Test pH, [salt], perhaps metals.	Start with quick and dirty
tests of something like a dotblot apparatus.  Do digests of the labelled DNA
to try to isolate bound signal.  You could also try these tests as only the
incubation stage, and run the parallel trials on your current gel.  Maybe try
Tris-Borate buffer to start, but you will only need in the gel.  In theory
the EDTA only helps for shelf life(?).	Good luck, Warren


In article <3888D609.76B823A1 at capsidiol.ca.uky.edu>,
  Mark Schoenbeck <mark at capsidiol.ca.uky.edu> wrote:
> I am preparing to look at protein binding to regions of promoter DNA.  I
> would appreciate hearing from anyone who can suggest a systematic method
> for optimizing protein binding conditions for a gel mobility shift
> assay.  A previous lab member here used nuclear and whole-cell extracts
> and allowed 10 minutes for binding before running the labeled DNA
> through a 12% acrylamide 0.5 x TBE buffered gel.  Can anyone tell me if
> this is even a suitable buffer for retaining protein-DNA interactions?
>
> Thanks in advance for any assistance you can offer,
>
> Mark Schoenbeck
> Agronomy
> University of Kentucky
>
>


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