| Assay Method Information | |
| | In Vitro Assay |
| Description: | Human recombinant HDAC family proteins with His and GST tags were expressed using insect baculovirus expression system, and a biologically active HDAC family recombinant protein was obtained through Ni affinity column protein purification. HTRF detection method was used with H3(1-21) K9Ac biotin as the substrate. HDAC protein, gradient diluted compounds and the substrate were added to a white 384-well flat-bottomed microplate (ProxiPlate-384 Plus, PerkinElmer) and reacted for 1 hour, and a mixture of Eu labeled H3K9 antibody and XL665 labeled streptavidin were added and equilibrated for 0.5 hours at room temperature. By using the principle of HTRF, time-resolved fluorescence at 615 nm and 665 nm was detected using a microplate reader, and the ratio therebetween was calculated, and the corresponding enzyme activity inhibition rate was calculated by using GraphPad for analysis. Briefly, 20 μl of the activity testing system included the HDAC substrate (0.2 μM. 4 μl), the Human Recombinant Protein HDAC (2-5 ng)/μl. 4 μl), the compound (2 μl), a mixture (10 μl) of Eu labeled H3K9 antibody and XL665 labeled streptavidin, and all components were diluted with Tris buffer (50 mM Tris-HCl, pH 8.0, 137mM NaCl, 2.7 mM KCl, and 1 mM MgCl2). |
| Affinity data for this assay | |
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