Immunoprecipitation conditions

Unfortunately, the conditions for the immunoprecipitations (especially the stringency of the binding and wash buffers) have to be determined experimentally for each antibody. These stringent conditions, on the one hand, can help to reduce the background, but on the other hand, can result in a loss of signal. 

The first step in the biochemical analysis of the receptor, isolation from the cell membrane, was difficult the membrane-associated protein is present in only small amounts and is insoluble in aqueous solution. Sensitivity and selectivity would be improved by radiolabeling under conditions that maintain cell viability so that only proteins associated with the surface and not those within the cell become labeled. A procedure that met these conditions was iodination catalyzed by lactoperoxidase in the presence of hydrogen peroxide (Marchalonis, 1969 Phillips and Morrison, 1970 Marchalonis et al., 1971). It was also necessary to find conditions for solubilizing membrane proteins that would not interfere with subsequent specific immunoprecipitation. 

In the second procedure, it is possible to assay specific immunoprecipitated proteins (e.g., PI 3-kinase subunits, receptors, receptor component chains, or even cellular proteins) for associated lipid kinase activity under in vitro assay conditions using distinct substrates such as Ptdlns (6-8). It is also possible to use PtdIns(4)P or PtdIns(4,5)P2 as in vitro substrates for the prototypical class IA PI 3-kinase, but generally these are more expensive to buy. 

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]. 

One of the earliest applications of immunochemical techniques in the field of molecular biology was the immunoprecipitation of an enzyme as a means of demonstrating its de novo synthesis. Such a demonstration in bacteria might involve growing the cells under two sets of physiological conditions, one in which the enzyme is expected to be present and a second in which it is not. A radioactive amino acid is added to each culture as a means of labeling newly synthesized proteins. The cells are... 

We have attempted to select conditions that minimize the effects of sample compositional differences. The efforts have included using EDTA as part of the assay buffer, using sufficient reaction time, optimizing for maximum sensitivity, which permits dilution of the sample, elevating the concentrations of nonimmune y-globulin and second antibody, and, in some cases, rejecting a specific lot of second antibody. The time required for completion of immunoprecipitation using low concentrations of reactants can require up to a week at 4°. However, use of sufficient concentra-... 

The location of individual proteins or protein subunits has been established by analyzing pure proteins and immunoprecipitates prepared using specific antisera, or by co-electrophoresis. Proteins containing cysteine residues can be specifically labeled using [ I]iodoacetamide and can be located autoradiographically. If analysis is performed under nondenaturing conditions, specific stains are available to locate certain types of protein, e.g., glycoproteins (A14, H12) and enzymes. 

---