kinase - kinase activity (Nov/29/2006 )

hi

if I want to measure a kinase activity of a a kinase.
what is the best way?

thanks

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you have to distinguish between cis(auto)phosphorylation and trans(substrate)phosphorylation; what you actually do depends on how much money you like to spend. You can buy kits f.i. as ELISA, or really expensive FRET solutions for substrate phosphorylation; the classic is to radioactively phosphorylate basic peptides which can be bound to phosphocellulose, washed in phosphoric acid and counting (Roskoski protocol, see Methods Enzymol 1983 I think) cheaper but you have radioactivity...

autophosphorylation can be monitored with appropriate phospho-specific Ab if available, or again with radioactive gamma-32P-ATP...

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there is a FRET technique to check the kinase activity??

FRET is for interaction analysis.
right


you have to distinguish between cis(auto)phosphorylation and trans(substrate)phosphorylation; what you actually do depends on how much money you like to spend. You can buy kits f.i. as ELISA, or really expensive FRET solutions for substrate phosphorylation; the classic is to radioactively phosphorylate basic peptides which can be bound to phosphocellulose, washed in phosphoric acid and counting (Roskoski protocol, see Methods Enzymol 1983 I think) cheaper but you have radioactivity...

autophosphorylation can be monitored with appropriate phospho-specific Ab if available, or again with radioactive gamma-32P-ATP...
[/quote]

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eg.

develop an assay such that u have a C14 labelled substrate for the kinase and then mix them together and then using a scintillation counter, get the activity.

U need to have extracts where ur kinse is abundant or purifired protien or extracts from transient transfection.

or u could try to develop an spectrophotometer assay.

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FRET analysis for kinase activity use peptide or protein substrates which carry at the opposite ends fluorochromes, mostly a CFP/YFP sytem; phosphorylation alters the conformation which can be monitored by FRET

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Well I do FREt for interaction purpose.
I don't see how you do FRET in this case.

let's see you want to see if the protein ...ADFYNKVS... is phodpotylated at ...ADFpYNKVS...

you will need to have CFP...ADFYNKVS...YFP.
the problem is that you will FRET because of dimerization and specific interaction between CFP and YFP from the N and C terminal

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Invitrogen provides several more or less specific kinase substrates utilzing FRET, product name is Z´Lyte; it works in vitro; for in vivo, you need expression vectors for specific FRET substrates; it is VERY difficult to tune the parameters, I have tried

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