Liquid HAT Assay
Method
20-µl Reaction
- 4 µl 5X HAT Buffer
- 13.2 µl Water
- 1 µl Enzyme
- 0.8 µl 3H-acetyl CoA
- 1 µl 10 mg/ml Histones or Substrate
- Set up reaction in order listed.
- Incubate 30 minutes 30°C. Spin down briefly.
- Spot half of reaction on Whatman® P81 phosphocellulose filter paper circle and air dry.
Note: Prelabel filter papers with pencil on top of Saran Wrap®. - Wash filters for 30 minutes at 4°C in ice-cold 10% trichloroacetic acid (TCA).
- Wash 2X, 5 minutes each, RT, in 10% TCA.
- Wash 1X 5 minutes in 100% ethanol. Let filters dry under heat lamps.
- Place in scintillation fluid and count 3H.
- When appropriate, you can electrophorese other half of reaction.
- Add 2X SDS-PAGE buffer
- Boil
- Load on 22% PAGE gel (60:0.4 acrylamide:bis) resolver with standard stacker. Run medium sized gel approximately 1700 V/hr. Stain with Coomassie and destain. Then fluorograph by placing in EN3HANCE for 30 minutes, then water, for 30 minutes. Dry on heated gel dryer. Develop at -80°C for 3-5 days to see which histones/proteins have been labeled.
5X HAT Buffer
- 75mM Tris, pH 7.8
- 1.25mM EDTA, pH 8.0
- 0.25% Tween-20
- 12.5mM DTT
- 25% Glycerol
Protease Inhibitor Cocktail
- 10 µg/ml PMSF
- 1µM Pepstatin
- 1µM Leupeptin
- Add 1 µl per ml protein sample
*This procedure is basically as described by Brownell and Allis, PNAS 92, 6364-6368, 1995.