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SIZE OF PHOSPHORYLATED PROTEINS - (Jan/20/2006 )

Hi!
This is my first time to use this question/answer session. I want help with phosphrylated proteins. Do thet migrate at a different rate than the unphosphoryalted form when bothe are run of the exact same type of SDS_PAGE gels?
Please help?
I am ging cross-eyed looking at my gels trying to see if my protein got phosphorylate.

Thanks to 1 and all
Mushu

-Mushrooknob-

hm ... phosphorylation means a very small change in mobility. in general you'll never be able to see the state of phosphorylation in a normal SDS-PAGE to my knowledge

-Kersten-

I think if you're careful, you can see phosphorylation if you have one of those large gel boxes (ie not BioRad Mini cell).
If there's a phospho antibody for your protein it would probably be much easier to use that instead though.

-Mountainman-

Depends on the rate of phosphorylation. Some proteins show differences in mobility that are easily seen... Are you doing WB or just staining the gel? If WB, you can use some phosphatase to dephosphorylate your protein and check for any difference in mobility.

There are some commercialy available antibodies that detect phosphoylation, but usualy only work on purified proteins. You'd see only a huge smeer on extracts.

And I also have the feeling that MS could answer your question, but again you need to purify a small amount of your protein.

-UniSPheryk-

QUOTE (UniSPheryk @ Jan 20 2006, 08:21 PM)
Depends on the rate of phosphorylation. Some proteins show differences in mobility that are easily seen... Are you doing WB or just staining the gel? If WB, you can use some phosphatase to dephosphorylate your protein and check for any difference in mobility.

There are some commercialy available antibodies that detect phosphoylation, but usualy only work on purified proteins. You'd see only a huge smeer on extracts.

And I also have the feeling that MS could answer your question, but again you need to purify a small amount of your protein.


the anti-pTyr Ab from the 4G10 clone is very efficient on Westren blotting of total cellular extract...

S├ębastien_

-tryptofan-

hello,

I worked a lot with phosphorylation.
I was doing a simple 8% SDS-PAGE, looking for proteins around 40 kDa,migrating at 20 mA until dye reaches the bottom of the gel, and I've never seen a difference in migration in such conditions.
however, there was papers showing it (once upon a time). I would say, migrate on a big gel, for a long time. choose very carefully the percentage of acrylamide.
but the easiest is to use anti-phospho antibodies like 4G10 or even better if you are lucky : an anti phospho specific. now there are more and more antibodies directed against the phosphorylated form of a cognate protein on the market. And this is what is published now.

-laurence-

we routinely run 10% SDS-PAGE for a 58kDa protein with autophosphorylation/dephosphoylation properties and can see the difference between the two species. one paper with the clearest pictures of this are:

Nishiwaki, T. et al "Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942." PNAS (2004) 13927-13932.


specifically look at the gels on page 13929

hope this helps!

-chempilot-