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Problem in immunoprecipitation


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#1 yjokim

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Posted 28 May 2004 - 12:15 PM

I got a problem with my I.P. I'm trying to immunoprecipitate a FLAG tagged protein from a transgenic worm lysate (total lysate) using anti-FLAG Ab-ProteinG-sepharose. When I analyze equivalent amounts of total lysate before IP, bead extract, total lysate after IP through western blotting, almost all (at least 90%) the tagged protein bands were disappeared in lysate after IP, while strong in lysate before IP. However I couldn't see ovbious band either in the bead extract. My question is 'Where is the disappeared bands?'

#2 phdconsult

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Posted 30 May 2004 - 10:19 AM

Your IP worked but the target protein has been rendered insoluble by the procedure. Add a chaperone like DnaK (Sigma Chemical Co) to see the disappeared bands again.




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