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In-Gel HAT Assay

Method

  1. Pour an 8% SDS-PAGE gel containing 1 mg/ml calf thymus histones (Sigma type IIA).

    Stacking gel can contain histones or not.

    Use a 10 mg/ml stock of histones dissolved in water, and replace part of the water portion of the gel with the histones. Add SDS last (after histones but before accelerants). The gel suspension should be slightly cloudy.

  2. Dissolve samples (potential HATs) in SDS-PAGE sample buffer with protease inhibitors, but do not boil.

  3. Following electrophoresis, wash gel for 1 hour in Buffer B with 20% isopropanol. Then wash for 30 minutes in Buffer B alone.

  4. Incubate gels in Buffer B containing 8M urea for 1 hour at RT, with agitation. The gel will swell slightly.

  5. Renature: Change to 50 ml Buffer B with 0.04% Tween-40, swish and incubate at 4°C, with agitation, for 30 minutes.

  6. Change to 50 ml fresh Buffer B with 0.04% Tween-40 and incubate O/N 4°C, with agitation.

  7. Wash in Buffer A for 30 minutes at 30°C. Then incubate with 10 µCi 3H-acetyl CoA in 10 ml Buffer A for 30 minutes at 30°C, with agitation. This works well if you use a Seal-a-Meal® bag.

  8. Wash gel extensively (5X, 1hour each) in 5% TCA, then wash O/N in 5% TCA, to remove unbound label.

  9. Fluorograph and expose to film 2 days to 2 weeks.

Solutions

Buffer A (100 ml)

  • 5 ml 1M Tris, pH 8.0
  • 0.1 ml 1M DTT
  • 0.02 ml 0.5M EDTA
  • 10 ml Glycerol
  • 0.1 ml 1M PMSF

Buffer B (100 ml)

  • 5 ml 1M Tris, pH 8.0
  • 0.1 ml 1M DTT
  • 0.02 ml EDTA

© 2014 The University of Texas MD Anderson Cancer Center