Immunoprecipitations

1. Prepare samples to immunoprecipitate at a final concentration of 1 mg/mL protein in equivalent volumes (supplement with lysis buffer as required). For immunopre-cipitation of IR-b, samples containing 200 ig protein work well. For IRS-1 or IRS-2, 500 ig protein or 1 mg protein per immunoprecipitation, respectively, is required for detection.

2. Add appropriate antibody. Generally, 0.5 ig anti-IR-b, 4 ig anti-IRS-1, or 10 ig anti-IRS-2 antibodies from the sources described in Subheading 2.2. work well to immunoprecipitate these proteins effectively. Rotate the tubes 1-2 h at 4°C (see Note 3).

3. Capture the immune complexes with the addition of 30 iL protein-A-agarose. Rotate for at least 30 min at 4°C.

4. Pellet the agarose beads with a brief 10-s spin in the refrigerated microfuge. Remove and save a small aliquot of the supernatant. Then, aspirate the remaining supernatant and wash the protein-A-agarose beads with 0.5-1 mL lysis buffer. Repeat two more times. When removing the buffer from the final wash, use a 1-cm3 syringe needle (26G5/8) attached to the aspirator to remove all of the wash solution from the beads.

5. Elute the immune complexes from the beads with the addition of 30 iL Laemmli sample buffer. Vortex well and boil the samples in Laemmli buffer 3-5 min. Pellet the agarose beads by centrifuging in a microcentrifuge for 1 min at maximum speed, at room temperature.

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