Immunoprecipitation under disruptive condition

Anal3Tsis of physiological interactions by inununopredpitation 292 Immunoprecipitation under disruptive conditions 292 Troubleshooting high background signals 292... 

Immunoprecipitation is performed under non-disruptive conditions to maintain physiological interactions. Protocol 14B can be used to recover and examine the subunit composition of intact oligomeric complexes containing the antigen and other associated macromolecules. The antigen protein is identified in the complex by subsequent immunoblotting or by performing immunoprecipitation imder disruptive conditions (Protocol 14C). 

In Protocol 14C, immunoprecipitation is performed under disruptive conditions in a mixed micelle detergent buffer (RIPA). Most physiological interactions are dissociated imder these stringent conditions, and usually only the antigen protein and veiy tightly-associated proteins are recovered by immunoprecipitation. This method is useful to quantitate levels of pS]methionine labelled protein antigen, and to study synthesis and degradation rates by pulse chase analysis. 

As with other in vivo selections, it is critical to validate hits at the end of the selection experiment with a secondary screen. The most common secondary screen used with the Y2H assay is a lacZ screen. If either the DBD or AD fusion is under control of an inducible promoter, this screen can be carried out both under inducing and non-inducing conditions to ensure that transcription activation is protein dependent. Y3H systems, where transcription activation depends on a bridging RNA or small molecule, and reverse Y2H systems, where a third protein is disrupting the interaction, provide a built-in control. One can simply check that transcription activation is in fact dependent on the third component. As with any in vivo selection, the evolved plasmid should be isolated and retransformed into a fresh yeast selection strain to ensure that the phenotype is plasmid-dependent. Ultimately, the interaction will be confirmed with co-immunoprecipitation experiments or other in vitro binding assays [39]. 

To immunoprecipitate proteins from embryonic tissue or cell culture, the material has to be disrupted under conditions that solubilize the proteins (see Notes 3 and 4). 

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