protein-protein interaction
Hasina Akhter
Saturday, 21 July 2007 08:27 UTC
Hello
i have found interaction between two proteins in yeast two-hybrid analysis.But i have not yet successful in proving this interaction by other methods i.e, co-immunoprecipitation technique.
is there any cheap and good method to prove this interaction? can anybody help me?
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Replies
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Lots of good protocols for analyzing protein:protein interactions here
In particular, if you’re looking for co-immunoprecipitation, try this:
Identification of Associated Proteins by Coimmunoprecipitation -
Dear Style
Thank u very much for ur reply.
Unfortunately I cant get access into Cold spring harbour protocol online.
Is it possible for u to send me the PDF version of the article on co-immunoprecipitation “Identification of Associated Proteins by Coimmunoprecipitation”?
I am very sorry if I bother u much.
regards
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I’d love to help, but that would be an infringement of copyright and against the law.
The protocol is available in Molecular Cloning 3rd Edition (popularly known as “Maniaitis”). If your lab doesn’t have a copy, ask around, I’m sure a lab somewhere near yours will.
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Hi Hasina,
Dorothy and I searched our freely available ‘Protocols Network’ and found these three protocols that might be of interest to you:
1) Immunoprecipitation of endogenous MEKK1 and kinase assay
- despite its very specific sounding title this looks to be a more generally applicable IP protocol.3) Efficiency of protein-protein interactions in mammalian cells determined by a dual-light assay
- we’re not sure how quick or cheap this last one is but it might be worth considering too.If you do try any of these protocols and you have any feedback then please do comment on them as your feedback might be helpful to later users. As these are in our ‘Protocols Network’ they’ve not been peer reviewed or edited, but uploaded direct by scientists. If you have any protocols of your own to upload, please do!
I hope this helps and good luck with the project,
Katharine -
Dear Katharine and Dorothy
Thx very much for sending those protocols.
If I use any of these protocls I will surely let you know the results.
best regards
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I have successfully used SPR binding assay to confirm and characterize protein-protein interaction identified by y2h. see my two recent papers: J Biol Chem. 2009 Jan 30;284(5):3227-38, J Biol Chem. 2009 Jan 16;284(3):1364-72, and a protocol: Methods Mol Biol. 2009;493:323-43. hope this will be useful.
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Hi Akhter
Yeast 2 hybrid (screening process) results should be confirmed by Co immunoprecipitation which you are trying for. But the problem is most of the y2h results are false positive, by saying that i don’t mean what you are doing is wrong. Probably you need to check the buffer you are using, if you are using RIPA buffer check the pH of Tris HCl. Because ph is also one of the factor which may disrupt the interaction.
Good luck
Sanjib
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