- Dilute the cell nuclear extract before beginning the immunoprecipitation to roughly 1mg/mL total cell protein in a microcentrifuge tube with PBS.
- Add 4 μg of ant-Myc Tag, clone 9E10 to 500 μg-1 mg cell lysate.
- Gently rock the reaction mixture at 4 ̊C for 2 hours or overnight.
- Capture the immunocomplex by adding 30 μL (15μl packed beads) of washed Protein G agarose bead slurry, catalog #16-266.
- Gently rock the reaction mixture at 4 ̊C for 2 hours.
- 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.
- Resuspend the agarose beads in 50μl 2x Laemmli sample buffer.
- 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.
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