Immunoprecipitation

1. Perform immunoprecipitations on lysates normalized to contain equal quantities of cellular protein. Aliquot lysate containing approx 300 ig protein into a labeled microcentrifuge tube and then dilute with lysis buffer containing protease inhibitors to bring the lysate up to a total volume of 500 iL.

2. Start the immunoprecipitation by adding the amount of antibody suggested by the manufacturer for immunoprecipitation. Rotate the tubes at 4°C for at least 1 h.

3. For each treatment condition, aliquot 50 iL of a 1:1 slurry of protein-A conjugated to agarose beads (or secondary antibody conjugated to agarose beads) into a new labeled microcentrifuge tube.

4. Wash the beads three times with 1 mL of ice-cold lysis buffer containing protease inhibitors. Pellet the beads by centrifuging in a benchtop picofuge after the addition of each wash. After the last wash, remove most of the fluid, leaving a very small volume above the beads.

5. Add the cell lysates containing the primary antibody to each of the new tubes containing the agarose-conjugated protein-A. Rotate this mixture at 4°C for at least 30 min.

6. During the incubation with the protein-A, thaw out the 20X kinase buffer. Prepare at least 3 mL of 1X kinase buffer and 25 iL of 1.5X kinase buffer for each reaction being performed.

7. After the 30-min incubation is complete, pellet the beads by centrifuging at 6000g for 30 s. Remove the supernatant.

8. Wash the beads three times with ice-cold lysis buffer containing protease inhibitors, then three times with 1X kinase buffer. Use approx 1 mL for each wash. Completely remove all liquid after the last wash (see Note 5).

9. Add 20 iL of 1.5 X kinase buffer to the beads.

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