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Elute immunoprecipitated complex with protein G?


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#1 fakeopal

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Posted 26 April 2010 - 07:34 AM

I want to immunoprecipitate a kinase and test its activity in vitro. I can successfully IP the kinase using protein G agarose beads. Based on the kinase assay performed on beads, the IPed kinase is active. Now I want to elute the kinase off the beads and screen for downstream targets. I'm worried that the usual elution conditions (low pH, etc) would disrupt the kinase activity. Alternatively, I could tag my kinase and elute with a peptide. Another approach I'm considering is to elute with protein G. I haven't seen any available protocol for this elution method. I'm wondering if anyone has used this approach and how well it works?

Any advice/help would be greatly appreciated!! Thanks!

#2 mdfenko

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Posted 26 April 2010 - 09:36 AM

you can elute with low pH into a tube with buffer to neutralize the solution (we use 1/5 volume of 1M phosphate pH 8). that way the protein won't be at the low pH long enough to cause any problems.
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