in vitro phosphorylation - (May/11/2010 )

Hi All,

Can anybody recommend me a non-radioactive in vitro phosphorylation assay, please?

Thanks!

tiramisu on May 11 2010, 10:48 PM said:

method, okay, but the aim of your examination is important; what do you like to examine?

Inmost sun on May 12 2010, 01:58 PM said:

Hi,

I would like to phosphorylate my recombinant protein and after examine what are the influences of this modifiaction on the enzyme activity, etc.

Thanks for your answer.

tiramisu on May 13 2010, 05:11 PM said:

you need a protein kinase which uses your protein of interest as a substrate; dedicated phosphorylations are prefered;

you can run a phosphorylation of your protein of interest with "cold" ATP, then run the enzyme assay of your protein;

for monitoring phosphorylation you may run a Westernblot, and use phospho-specific antibodies

you can use atp-gamma-s instead of atp. this will form a more stable, phosphatase resistant, phosphorylation (it will give you more time to work with the modified enzyme). the kinase will work a little slower so you will have to incubate a little longer.

you can monitor phosphorylation with urea-page (not sds), depending on the size of your enzyme.