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Immunoprecipitation Protocol

  1. Dilute the cell nuclear extract before beginning the immunoprecipitation to roughly 1mg/mL total cell protein in a microcentrifuge tube with PBS.
  2. Add 4 μg of ant-Myc Tag, clone 9E10 to 500 μg-1 mg cell lysate.
  3. Gently rock the reaction mixture at 4 ̊C for 2 hours or overnight.
  4. Capture the immunocomplex by adding 30 μL (15μl packed beads) of washed Protein G agarose bead slurry, catalog #16-266.
  5. Gently rock the reaction mixture at 4 ̊C for 2 hours.
  6. Collect the agarose beads by pulsing (5 seconds in the microcentrifuge at 14,000 x g) and drain off the supernatant. Wash the beads 3x with either ice cold cell lysis buffer or PBS.
  7. Resuspend the agarose beads in 50μl 2x Laemmli sample buffer.
  8. The agarose beads can either be frozen for later use or suspended in Laemmli sample buffer and boiled for 5 minutes. Collect the beads by a microcentrifuge pulse. SDS-PAGE and subsequent immunoblot analysis can be performed on a sample of the supernatant.