On Wed, 21 Jan 1998 12:22:25 -0500, 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.
>>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
steps).
HTH
Loïck Le Dantec
Lab. Biol. Mol. Cell
INRA-Bordeaux
loick.ledantec at hol.frledantec at bordeaux.inra.fr
