IUBio

staining gel shift gels

Peter Pediaditakis pxpst2 at vms.cis.pitt.edu
Wed Jan 21 15:21:49 EST 1998


In article <34C62ED1.4625 at niehs.nih.gov>, Mara Casar <casar at niehs.nih.gov>
wrote:

> I'm curious to see if anyone has run a gel shift gel and then stained
> for protein.  I'd like to get an estimation of my protein's molecular
> weight, but it's nowhere near pure (that's the rest of my project...) so
> I can't find it on SDS-PAGE.  What I would like to do is let my samples
> bind to the 32P-labeled probe, run them on a 4% acrylamide gel (0.5x
> TBE), and then stain the gel before exposing it to film.  I know that
> both Coomassie and silver staining protocols fix the gel before
> staining, but will this keep the DNA probe bound to proteins?  
> 
> Also, has anyone used molecular weight markers on gel shift gels?  I
> recently found some (Pharmacia) that are for use on "native" gels, which
> they say are Tris-glycine, no SDS.  Any ideas about whether they will
> run into a TBE gel?  The pH is about the same, so that shouldn't be a
> problem.

I am guessing but I think that you will have problems estimating the
Molecular weight of the protein causing the shift.  With gel shifts, the
gels are non reducing.  Proteins that have not been reduced will have a
tertiary structure that will give you a gross underestimation of the
actual MW.  This will happen for sure with your protein because it is a
DNA binding protein which are generally very folded proteins(in other
words condensed).  Also, a 4% PAGE gel will not give you the resolution
needed unless your protein is bigger than 200kD.
If I were you, I would try to run you gel shift as normal and cut out the
band of interest(shifted band) and electroelute the protein from the gel
and then run it on a 8-10% PAGE gel.  Then stain with slver.  
Hope this helps
Peter

-- 
"Don't you eat that yellow snow
    WAtch out where the Huskies go"

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