Detection of phosphorylation site - (Jul/23/2010 )
TimUCR on Tue Aug 10 19:26:16 2010 said:
Thanks for the suggestions everyone. There aren't phospho-specific antibodies for chloride channels as far as I can tell. I would prefer to just see if it can even get phosphorylated before I send it off to a mass spec lab. Instead of doing the kinase assay with the phosphate isotope, could I do a gel shift assay on SDS-PAGE instead? If the protein gets phosphorylated it should run slower? Will this be significantly noticeable or would it just be easier to do the kinase assay?
If the protein runs slower (not always the case...) then you can treat your sample w/o phosphatase and check that what you are seeing is really phosphorylation (and not sumoylation, for example...)
-laurequillo-
Is it likely that there won't be any mobility shift?
-TimUCR-
i used to do gel shift on urea-page (no sds or reducing agent). my protein was 20 kDa.
-mdfenko-
TimUCR on Tue Aug 10 21:02:12 2010 said:
Is it likely that there won't be any mobility shift?
It depends on your protein and the phosphorylation. I guess the up shift depends on the number of sites, and the percentage of protein phosphorylated.
Sometimes I saw some proteins phosphorylated by protein X in one site with and upshift and by protein Y in another site and no upshift.
-laurequillo-
with urea-page we saw a downshift of the phosphorylated protein (due to increased charge).
-mdfenko-
mdfenko on Wed Aug 11 19:37:52 2010 said:
with urea-page we saw a downshift of the phosphorylated protein (due to increased charge).
Ah ok! So with an Urea-page you should be able to see always a change in the mobility, is that right? (when you compare phosphorylated vs non-phosphorylated)
-laurequillo-
laurequillo on Wed Aug 11 19:54:39 2010 said:
Ah ok! So with an Urea-page you should be able to see always a change in the mobility, is that right? (when you compare phosphorylated vs non-phosphorylated)
yes. but you may have to work to find the proper acrylamide concentration for the protein that you are working with (we used 7.5% for the 20 kDa protein, i don't remember what concentration we used for 200 kDa protein)
-mdfenko-
mdfenko on Wed Aug 11 20:05:37 2010 said:
laurequillo on Wed Aug 11 19:54:39 2010 said:
Ah ok! So with an Urea-page you should be able to see always a change in the mobility, is that right? (when you compare phosphorylated vs non-phosphorylated)
yes. but you may have to work to find the proper acrylamide concentration for the protein that you are working with (we used 7.5% for the 20 kDa protein, i don't remember what concentration we used for 200 kDa protein)
That is good to know.
-laurequillo-
And Mdfenko, even if you have a single phosphorylation site, you can see the change in the overall charge?
-laurequillo-
This is all good stuff. Would using a combination of Urea-SDS-Page work better? My protein is ~75kDa, any suggestions on what concentrations I should be using?
-TimUCR-