Kit contains nonradioactive components for radiolabeling and purifying histone H4 peptide with [3H]-acetyl CoA. The radiolabeled peptide is a suitable substrate for assay of HDAc (histone deacetylase) activity. HeLa nuclear extract, which is rich in histone deacetylase activity, is included as a positive control. The HDAc inhibitor sodium butyrate is provided to demonstrate specificity of the deacetylase activity.
Quantity: Approximately 100 assays per 100ug of labeled peptide (the exact amount will vary according to labeling efficiency). Use of 5000-10,000 CPM of [3H]-acetyl histone H4 for each assay is recommended.
Quality Control Testing and Research Applications:
[3H]-acetylation of Histone H4 peptide: 70% of [3H]-acetate was incorporated into 100ug Histone H4 peptide
Experiment 1: Dependence of [3H]-acetate release (HDAc Activity) on the amount of HeLa Nuclear Extract. Increasing amounts of HeLa nuclear extract were incubated at room temperature with [3H]-acetyl-Histone H4 peptide (10,000CPM). Released [3H]-acetate was measured using a scintillation counter. HeLa nuclear extract is a source of HDAc activity.
Experiment 2: Inhibition of [3H]-acetate release (HDAc Activity) by Sodium Butyrate [3H]-acetyl-Histone H4 peptide was incubated overnight at room temperature with 20ug HeLa nuclear extract in the presence or absence of Sodium Butyrate. 250mM Sodium Butyrate inhibited release of [3H]-acetate by ~96%.
Storage and Stability: After receipt, store individual components at temperatures recommended above and on component labels. This kit is stable for 6 months from date of shipment.
1. Miyashuta, T., et al., FEBS Let. 353: 225-229, 1994.
2. Ohno, Y., et al., Proc. Natl. Acad. Sci. USA 94: 10279-10284, 1997.
3. Dignam, J.D., et al., Nucl. Acids Res. 11: 1475-1489, 1983.
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