Immunoprecipiation

Immunoprecipitation is a technique that allows the biochemical characterization of protein from relatively few cells, and is an important technique where material may be a limiting factor. It can also be used to study various types of posttranslational modification (see Note 8). The amount of material required will depend on the abundance of the antigen (see Note 9).

1. The lysates are precleared twice for 1 h at 4°C with 25 |L of protein A-Sepharose beads (see Note 10).

2. The precleared lysates are then immunoprecipitated with 10 ||L of 2 mg/mL antibody protein A-Sepharose beads for 4 h to overnight at 4°C.

3. The beads are washed six to eight times in lysis buffer for 1 min with as much of the solution as possible being removed at each wash (Note 11).

At this stage, the immunoprecipitate can be analyzed by standard fractionation techniques, such as SDS-PAGE, or assessed for enzyme activity (e.g., kinase activity).

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