Mononucleosome binding of GST-PHD: detection by Western blot

1. Add 200-400uL of Binding buffer to a 1.5mL microfuge tube.

2. Add 1 μG of purified mononucleosomes

3. Add 10 μG of GST-fused protein (whole protein, domain, etc.)

4. Incubate 4C O/N on rotating platform.

5. Add 30 μL of 50% Glutathione bead slurry.

6. Incubate 4C for 1 hour

7. Wash 3 times with 1mL of 1X binding buffer

8. Add 50uL of 2X SDS Sample loading buffer to the pelleted glutathione slurry.

9. Load 15-20uL per lane on a Western blot. Use antibodies against Histone H3 or any modification-specific antibodies of interest.

Binding buffer: 300 mM NaCl, 0.1% NP40, 50mM Tris 7.5, 10% glycerol