Immunoprecipitation

1.    Add 5-10 µg of antibody to the cold lysate (total volume of 500µl)

2.    Add 20 µl of Protein G slurry in prechilled Lysis Buffer

3.    Incubate for 2 hours at 4°C on the rotator

4.    Spin the eppendorf tube at 10,000xg for 30 seconds

5.    Carefully remove supernatant completely

6.   Wash the beads 3-5 times with 500 µl of Lysis Buffer

7.    Add 50 µl of 1X Laemmli sample buffer to bead pellet

8.    Vortex and heat to 90-100°C for 10 minutes.

9.    Spin at 10000xg for 5 minutes

10.  Collect supernatant and load onto the gel or freeze

Protease Inhibitor Cocktail (100X)

    * PMSF, 5 mg (50 µg/ml)

    * Aprotinin, 100 µg (1 µg/ml)

    * Leupeptin, 100 µg (1 µg/ml)

    * Pepstatin, 100 µg (1 µg/ml)

    * 100% Ethanol bring up to 1 ml, aliquot and keep at minus 20°C.