| Assay Method Information | |
| | Kat6a Activity Assay |
| Description: | Kat6a inhibitory activities of the compounds described in the present invention were quantified using a Fluorescence Resonance Energy Transfer (TR-FRET) assay which measures acetylation of a synthetic, biotinylated Histone-H4-derived peptide by the enzyme.Recombinant human His-tagged Kat6a protein (amino acids 194-810), was purified inhouse from Baculovirus- infected insect cells (Sf9). Histone H4 peptide (amino acids 1-24, SGRGKGGKGLGKGGAKRHRK-VLRD-K(Btn)-amide), which was used as substrate, was synthesized by Biosyntan GmbH, Berlin, Germany. Acetyl Coenzyme A was purchased from Sigma-Aldrich (#A-2056).Kat6a was incubated for 60 mins at 22°C in the presence of different concentrations of test substances (0 pM, and within the range 0.01 - 20 pM) in assay buffer [25 mM Tris/HCI pH 8, 1 mM EGTA, 2.5 mM Glutathion, 0.02% Chicken Albumin, 0.05% Pluronic F127, 25 mM NaCI, 220 nM H4 peptide and 600 nM Acetyl Coenzyme A],The reaction was stopped by addition of Detection Solution (25mM HEPES pH 7.5, 0.1 % BSA, 22nM SAXL665 (Cisbio #610SAXLE), 100pM Anacardic Acid (Enzo #ALX-270-381), 1 nM Anti-Histone H4 (ACETYL K8) Antibody (ABCAM #AB15823) and 0.5 nM AntiRabbit IgG Eu (Perkin Elmer #AD0083). |
| Affinity data for this assay | |
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