staining gel shift gels

loick.ledantec at hol.fr loick.ledantec at hol.fr
Thu Jan 22 11:48:46 EST 1998

On Wed, 21 Jan 1998 12:22:25 -0500, Mara Casar <casar at niehs.nih.gov>

>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
>Thanks in advance...
>Mara Casar

Another way to estimate molecular mass of your DNA-binding protein is
the southwestern analysis (see "The detection of DNA-binding proteins
by protein blotting. Nucleic acids research; Vol 8, p1-21; Bowen B. et
al;1980" for details). Proteins are separated on SDS-PAGE and
electroblotted to nitrocellulose membrane (like in western analysis).
The replica is used to detect DNA-binding proteins by incubation of
the filter with your 32P-labeled probe. It worked fine for me (i used
southwestern to detect a DNA-binding protein during the purification

Loïck Le Dantec
Lab. Biol. Mol. Cell
loick.ledantec at hol.fr
ledantec at bordeaux.inra.fr

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