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Immunoprecipitation vs. Affinity Chromatography For Eventual Mass Spec Analysis
Posted by: Pitkin (IP Hidden, New member, 1)
Date: August 5, 2005 10:02AM

We are trying to identify cellular proteins that interact with our viral protein of interest. We have chosen two biochemical approaches, one where we made monoclonal antibodies and IP'd viral protein from infected cells, and did mass spec analysis on binding partners. The other approach, which I am now attempting, is to His tag our viral protein and do affinity chromatography. I am in the process of writing a grant, and want to discuss the advantages vs. disadvantages of both approaches (keep in mind that we eventually want to identify binding partners by mass spec). Here are my questions:

What are the limits of immuprecipitation on protein yield (i.e. I know that more protein can be pulled down by affinity chromatograpy, as opposed to IP with a high affinity antibody, but how much more? estimated numbers in mg? 10X more? 5X more?) I assume this is ultimately an issue of how much cell lysate can be probed with antibody vs. how much can be poured over a column.

What are other advantages of using affinity chromatography over IP besides having more protein to analyze by mass spec?

Thanks in advance. I'm hoping that someone out there has debated these issues when trying to decide which protocol is best for identifying interacting partners of a protein (by Mass Spec).

 

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Re: Immunoprecipitation vs. Affinity Chromatography For Eventual Mass Spec Analysis
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: August 5, 2005 04:08PM

I would say the big advantage of using IP is its fast and simplicity. Mass spec analysis is so sensitive and requires so little proteins that makes the yield not a big issue, unless the less abundant protein of interest is badly contaminated with keratin.

 

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