In Vitro Kinase Assays - (Feb/17/2005 )

Hey everyone!
have been doing in vitro kinase assays using peptide substrates for a while now, they're all optimised and everything. Just wondering is there a way, apart from phosphate incorporation measurement, of telling whether the peptide has successfully bound the p81 membrane? A kind of ponceau stain equivalent?

Thanks!

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Hi Glow in the dark. How effective is the p81 assay? For example, do you use chemically produced peptides or stuff overexpressed in bacteria - personally, I will be using the latter.
What is the cost of those assays? I assume you already know the kinase. In my case, I am searching for the kinase. Thanks for listening and for any words of wisdom

As far as your Ponceau question, I unfortunately do not know. Good luck with it though.

passthrough

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you think of a positive control to evaluate binding efficiency of kinase substrate peptides

I often used the Roskoski protocol but never checked the binding efficiency for our kinase substrate peptides; if there was enough kinase activity, we had very high radioactive signals compared to background control, and were content; binding to P81 is under acidic conditions for basic aminoacidyls in your peptide; there may be an influence of incorporated phosphoryl to binding efficiency, so phosphosignals should actually even higher than counted;

for protein staining methods targeting the peptide bound you need high amount of peptide but substrate peptides are very expensive; moreover, P81 is not very stable for in aquatic solution; may be you succeed with colloid carbon (India Ink if you have)

or may be you have an antibody against your peptide;

If you will try those experiments to binding efficiency, I would be interested in your general findings

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Hi Passthrough,
The p81 assay worked well for me - I was using commercial peptide substrates (or sometimes proteins like histone to optimise the assay or as a positive control), never had the patience for the bacterial step!

The assays are pricey enough but for me the biggest cost was the peptides, which won't be an issue for you. I was testing out 5 different kinases, each with a different buffer so some of them (like GSK-3beta) were quite cheap, using reagents you'd have in the lab anyway while others like the PKC one was a bit more expensive as more specialised reagents had to be bought in. Besides that, it was just the cost of the p32 isotope and the recombinant enzymes which depends on where you're working and who your distributer is.

I did assays using PKC, PKC, CK1, CK2 and GSK-3beta - I can send you on the protocols I used if you like? Although if you're going to be screeening a load of kinases you might be better off sending your substrate to a company like Kinasource (lovely people in Scotland, very helpful) who'll screen a lot of kinases quickly and probably at a cost price which will be less than the price of reagents and time for you, especially if you run into any problems with the assays.

Best of luck with everything!

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Hey!
I stopped doing those assays last year because we managed to get the answer we were looking for in a different way - thanks for all your suggestions though. The reason I was looking for a way to measure binding efficiency was because if our substrate had been phosphorylated, the overall charge of the peptide would have been neutral or very slightly negative and I was wondering whether that was interfering with a positive signal I could have been getting as you said in your post - all my positive controls were very strongly postive you see,and we'd a good feeling about our little peptide so we looked at all possibilities. As you said, the amount of peptide substrate i'd have needed to detect binding to the filter was huge compared to the amount I had to work with so after a couple of tries we gave that approach up - although I never thought of an India ink approach.... ah well!

Thanks very much for your post, if I ever start those assays again in the future I'll let you know if I try any of your suggestions

All the best,
Glow In The Dark

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In response, I'd really appreciate those protocols. And, at Kinasource, do you have a contact. I can see them being helpful.
Thanks

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No need to thank with the protocols, I'm glad to pass them in if they're of use - will email them on once I find them!

We contacted Kinasource one day - just emailed someone we saw on the website (can't remember the specifics, it was nearly 2 years ago, sorry), all we said was what we were trying to do and with what. Within an hour some guy rang back saying that while they didn't provide the service that would have suited us but he spent about 15 minutes on the phone discussing the project, offering suggestions and giving references for papers we might have found useful - and all just because he was interested, not because we were clients or anything. Lovely man! The web-address is below

http://www.kinasource.co.uk

Will email those protocols onto you ASAP!

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I emailed them as well, but without the same response. In fact, there is no response yet, but it could be the time difference.

Take care

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You could try a Canadian company, Signalchem (http://www.signalchem.com/) if you are interested in screening. They offer inhibitor screening, so I suspect they'd do a substrate screen as well. I've e-mailed them about issues and they're always willing to help.

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I will check. Hope you read my other recent post and that it sparks an idea, of which you have had many already.

Thanks again

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