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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
  1. Set up reaction in order listed.
  2. Incubate 30 minutes 30°C. Spin down briefly.
  3. 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®.

  4. Wash filters for 30 minutes at 4°C in ice-cold 10% trichloroacetic acid (TCA).
  5. Wash 2X, 5 minutes each, RT, in 10% TCA.
  6. Wash 1X 5 minutes in 100% ethanol. Let filters dry under heat lamps.
  7. Place in scintillation fluid and count 3H.
  8. 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.


© 2014 The University of Texas MD Anderson Cancer Center