Immunoprecipitation of a secreted protein - (Jun/20/2007 )

Does anyone have a protocol for how to IP using cell culture media instead of using lysates as my protein of interest is secreted.

Please help!!!

Thanks!

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the protein conc of the medium would be much lower than of a lysate; you either add Ab in large amount to the medium, or try to concentrate your secreted protein before IP; most important are pH and ionic strength;

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Do you already know in what amounts your protein of interest is secreted. If it is very low, believe me you will have a tough time going for IP. One of the best method is that you concentrate it using concentrators?

Thanks

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Hi,

I've got some Amicon Ultra spin columns from Millipore which i have used before to concentrate cell lysates do u reckon if i used these first that would help. Not sure about how much of my protein will be present which doesnt help.

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Yes, I think they can be of real help. How much supernatant do you want to concentrate? You can also use large volumes and large 15ml columns to concentrate them.

thanks

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Thanks again, ill try using the filter and see what results i get, any idea on what concentration of Ab and Protein G/A i should use per ml of media?

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