Immunoprecipitation with immobilized protein

Monoclonal antibodies often do not bind well to protein A. To precipitate the antigen/antibody complexes anyway, you perform one more incubation with anti-mouse IgG (Promega, Jackson Lab), protein G sepharose (Sigma), or anti-mouse IgG sepharose before adding the immobilized protein A. 

An IP without control is like landing a plane without light. The following control experiments should be performed for an IP. 

Doolittle, M., et al. (1991). A Two-cycle Immunoprecipitation Procedure for Reducing Nonspecific Protein Contamination, Biochem. 195 364-368. 

(1986). Highly Sensitive Immunoadsorption Procedure for Detection of Low-abundance Proteins, biochem. 156 126-135. 

Sakamoto, J., and Campbell, K. (1991). A Monoclonal Antibody to the ()-subunit of the Skeletal Muscle Dihydropyridine Receptor Immunoprecipitates the Brain to-conotoxin GVIA Receptor, J. Biol Chem. 266 18914-18919. 

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Fig. 1. Immunoprecipitation of poly(ADP-ribosyl)ated cellular protein. (A) Nuclear proteins of human T-lymphoblastoid line CEM cells were poly(ADP-ribosyl)ated with [32p]NAD according to the method described before (4). Then, cells were lysed in NET-2 buffer ( 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA and 0.5 mM PMSF). Solubilized proteins were reacted for 2 hr with human serum IgG, either from control (C) or from patients (anti-poly(ADP-ribose) polymerase), which had been immobilized onto protein-A Sepharose CL-4B beads. After centrifugation and washing of the beads with NET-2 buffer, antibody-bound protein was solubilized in Laemmli s sample buffer and boiled for 2 min. Immunoprecipitated proteins were fractionated on a 12.5% polyacrylamide gel containing 0.1% SDS, and autoradiographed. (B) HeLa cell proteins were labelled with [ sjmetjiionine overnight, and solubilized in NET-2 buffer. Immunoprecipitation was processed as described above.

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