SDS-PAGE gel-shift to look at protein phosphorylation

Brian Hollander bahsel at sky.net
Fri Jan 16 19:09:41 EST 1998


My suggestion would be to vary the crosslinker concentration.  With
"long" gels, as opposed to mini-gels, I have achieved very good
resolution of proteins just slightly larger than those you mentioned 
using 10%T/0.8%C gels.

Martin Offterdinger wrote:
> 
> On Wed, 14 Jan 1998 12:14:40 -0800, Bob Steinberg
> <rsteinbe at etowah.ouhsc.edu> wrote:
> 
> >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
> I woulg suggest to use higher AA percentages to increase
> resolution(10-12%). A prestained protein marker would also be of help
> to avoid running out of your sample of interest. With 6-7 % or even 8%
> gels, the resolution in your MW range is quite limited!
> martin



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