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Immunoprecipitation of cell culture media

immunoprecipitation

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#1 jmafs

jmafs

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Posted 06 June 2012 - 10:40 AM

Hi everyone,

I've read on the forums about trying to immunoprecipitate proteins from cell culture media. I would like to do this to isolate a protein for western blotting while avoiding all the contaminants from the media serum. From what I've gathered so far is that I can concentrate the media using ultracentrifugation. From here, I am a little confused whether I can begin the immuno protocol and incubate with antibody, or if the sample needs further processing.

For cell lysates, there are a bunch of denaturing agents, salts, buffers, chelating agents, and protease inhibitors in the sample and it is also boiled. My question is, do I need to supplement the media to achieve the effect of a generic lysis buffer or is no further processing needed? Does the lysis buffer aid in antibody binding at all, or does it simply serve to isolate the proteins from the original cell culture?

Thanks!





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