This article describes the immunoprecipitation protocol:
- Wash protein A- or G-Sepharose or protein L-Agarose with 10X volume of RIPA buffer.
- Vortex and centrifuge for 1 minute in a microfuge.
- Resuspend the pellet in the original volume that the protein A or protein G matrix was in.
- Add protein A- or G-Sepharose or protein L-Agarose to the sample and incubate at 4°C for 30 minutes with shaking.
- Centrifuge for 1 minute in a microfuge to pellet absorbed nonspecific proteins and insoluble material. Discard.
Incubation with Primary Antibody
- Add primary antibody to precleared sample and incubate for 1 hour at 4°C with gentle agitation.
- Add protein-A or -G Sepharose beads and incubate 1 hour at 4° C with gentle agitation.
- Centrifuge for 1 minute in a microfuge. Wash the pellet in 1 ml of RIPA buffer.
- Repeat twice.
Removal of Antibody-antigen Complex
- Resuspend immunoprecipitate in a buffered solution with either 1% SDS and 15 mM beta-mercaptoethanol or 8M urea.
- Heat at 90-100°C for 5-20 minutes with occasional vortexing.
- Centrifuge for 1-2 minutes in a microfuge.
- The supernatant is ready to be analyzed by Western blot.
ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulins, primary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina