SDS-PAGE gel-shift to look at protein phosphorylation

Bob Steinberg rsteinbe at etowah.ouhsc.edu
Wed Jan 14 15:14:40 EST 1998


John Pawlowski wrote:
> 
> Hi all,
> I'm currently looking at the phosphorylation state of a protein by
> SDS_PAGE gel shift analysis. The protein is about 46 kDa and a noticible
> shift is seen using 8% polyacrylamide, 1.5 mm gel, approx 15 cm long.
> Unfortunately the sample is very near the bottom of the gel. I would like
> to increase the resolution of the gel-shift. Using 6 or 7% gels does not
> allow good separation of the protein from the dye front. Someone has
> suggested to me that I try running the sample longer using an 8% gel in a
> DNA sequencing gel apparatus (0.2 or 0.4 mm thick). Any thoughts on
> whether or not this approach would work or have any other ideas? ie.
> playing with the pH of the resolving gel, concentrations of cross-
> linkers? Any advice will be greatly appreciated. Thanks in advance.
> 
> John Pawlowski
> johnp at qadas.com

In my experience, the most important variable is pH of the lower gel--
we generally make a series of gels with lower gel buffer pH varying by
about 0.05 pH units between 8.6 and 8.9. Good luck.

Bob



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