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Help design a protein-protein interaction using co_IP


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#1 stagius24

stagius24

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Posted 26 November 2010 - 01:10 PM

Hello,

This is part of my undergrad project that involved studying a protein.
As part of my project, i want to study whether my protein will bind and acetylase core Histone or not. I have clone, purified the interested protein. I also had Histone H4,H3 in stock.

What I want to do is to design an experiment where my protein will bind to core histone by using co-IP approach. Here is my thought. Please comment.

Mix my protein with Histone in appropriate buffer
pull one protein with antibody, later with agarose bead
finally, detect with histone antibody
do a WB to confirm size change.

I did my control experiment first with no antibody binding first, but i got high background that will mask the result later. I would like everyone to comment on this traditional co-IP approach. Does it worth to buy the commercial kit or can I still improve the protocol somehow?

Regards,




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