Materials

1. Protein A- or G-coupled beads (e.g., protein A/G-coupled Sepharose 4 Fast Flow, Amersham; see Table 1 for species specificity of protein A or G).

4. Extraction buffer (EB; see Note 1 and Chapter 29): 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.05% Triton X-100, 1 mM dithiothreitol (DTT), 5 mM sodium P-glycero-phosphate, 0.5 mM sodium orthovanadate, 1 mM EDTA, 0.5 mM phenylmethyl-sulfonyl fluoride (PMSF), 10 ^g/mL aprotinin, 5 ^g/mL leupeptin, 2 ^g/mL pepstatin.

5. Rotating wheel.

6. 2X SDS sample buffer: 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol, 2 mM EDTA, 0.1 mg/mL bromophenol blue.

Fig. 1. The principle of the co-immunoprecipitation assay. Protein A/G-coupled beads are commercially available (see Heading 2). First, an antibody is attached to protein A/G-bearing beads. The antibody-coupled beads are then incubated with tissue extract. During the incubation, protein complexes containing the target antigen for the antibody are bound to the antibody-protein A/G-beads.

Fig. 1. The principle of the co-immunoprecipitation assay. Protein A/G-coupled beads are commercially available (see Heading 2). First, an antibody is attached to protein A/G-bearing beads. The antibody-coupled beads are then incubated with tissue extract. During the incubation, protein complexes containing the target antigen for the antibody are bound to the antibody-protein A/G-beads.

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