Detection of phosphorylation site - (Jul/23/2010 )
laurequillo on Wed Aug 11 20:16:21 2010 said:
And Mdfenko, even if you have a single phosphorylation site, you can see the change in the overall charge?
we were seeing differences with 1-2 phosphates.
also. we used atp-gamma-s instead of atp for a more stable phosphorylation (urea-page worked equally well with either).
-mdfenko-
TimUCR on Fri Aug 13 17:49:14 2010 said:
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?
do not use sds. urea enhances charge separation, sds gives equal charge density and would defeat the purpose of the urea. that being said, we have added urea to sds-page to help sharpen banding of high molecular weight proteins.
-mdfenko-
mdfenko on Fri Aug 13 23:58:21 2010 said:
laurequillo on Wed Aug 11 20:16:21 2010 said:
And Mdfenko, even if you have a single phosphorylation site, you can see the change in the overall charge?
we were seeing differences with 1-2 phosphates.
also. we used atp-gamma-s instead of atp for a more stable phosphorylation (urea-page worked equally well with either).
So, You did a kinase assay in vitro and then run the urea-page, And you detect your protein By staining the gel. (I am asking because I never used urea-page before)
-laurequillo-
laurequillo on Sat Aug 14 05:29:25 2010 said:
So, You did a kinase assay in vitro and then run the urea-page, And you detect your protein By staining the gel. (I am asking because I never used urea-page before)
yes. we were phosphorylating smooth muscle myosin light chains, running on urea-page and staining with coomassie.
-mdfenko-