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Immunoprecipitation is a widely used method to purify specific proteins from complex samples such as cell lysates or extracts. Conventional IP protocols use Protein A or G coupled to an insoluble resin, such as agarose beads, to capture an antigen: antibody complex in solution. The complex is then “precipitated” by centrifugation. Limitations of conventional IP include sample handling and processing difficulties, the inability to release native antigen from the beads for functional assays and poor reproducibility and recovery due to multiple wash steps.
This protocol is applicable only to kinases whose activity is not altered by cell lysis or immunoprecipitation procedures and that do not require soluble cofactors for activity. Please consult the following resources for additional information:
Verify that the antibody used quantitatively immunoprecipitates the kinase from lysates without affecting its activity, either positively or negatively.
Plan out the necessary components (antibody, buffers, isotope, substrate, and P81phosphocellulose cation-exchange paper/gel lanes) in advance by determining the number of reactions required. Plan on including negative controls (lysate of unstimulated cells; immunoprecipitation with non-immune antibody; sample with no enzyme) as well as a positive control (purified active enzyme). Establish conditions for the kinase assay to ensure that activity is measured well within the linear portion of the reaction (the limiting component should be the availability of active kinase, with substrate and ATP in excess). The most highly active samples are the first to deplete reactants, slowing activity in those samples, and resulting in a compression of measured activities between various samples.
The choice of lysis buffer is extremely important in any immunoprecipitation protocol. The lysis buffer must effectively solubilize the kinase of interest without denaturing it or altering its binding to antibodies. The use of nonionic and/or anionic detergents is used.
Nonionic detergents (e.g., Triton X-100, NP-40) break lipid/lipid and lipid/protein interactions. The anionic detergents (e.g., SDS, Sodium Deoxycholate) are denaturing than nonionic detergents and function to break protein-protein interactions. (Please consult: Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory.Cold Spring Harbor, N.Y.) As a general rule of thumb, to maintain kinase activity, do not use Na Deoxycholate, and for unstable kinases, do not use RIPA buffer EDTA should chelate all cellular Mg2+ and prevent phosphorylation. Phosphorylation can occur following cell lysis, since the cellular pool of ATP serves as the phosphate donor. The Na-vanadate will inhibit most tyrosine protein phosphatases and fluoride should inhibit some serine/threonine specific protein phosphatases.
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