SDS-PAGE gel-shift to look at protein phosphorylation

Martin Offterdinger a8803349 at unet.univie.ac.at
Thu Jan 15 03:20:52 EST 1998


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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