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in vitro phosphorylation


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5 replies to this topic

#1 tiramisu

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Posted 11 May 2010 - 02:48 PM

Hi All,

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

Thanks!

#2 Inmost sun

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Posted 12 May 2010 - 12:58 PM

Hi All,

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

Thanks!


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

#3 tiramisu

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Posted 13 May 2010 - 09:06 AM

Hi All,

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

Thanks!


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



#4 tiramisu

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Posted 13 May 2010 - 09:11 AM

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.

#5 Inmost sun

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Posted 13 May 2010 - 10:27 AM

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.


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

#6 mdfenko

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Posted 18 May 2010 - 10:40 AM

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.
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