| Assay Method Information | |
| | In Vitro Enzyme Inhibition Activity Assay |
| Description: | The purpose of this experiment was to test the in vitro inhibitory activity of the compounds against LSD1. The human recombinant LSD1 protein used in the experiment was purchased from Active Motif, and the enzyme level activity testing assay of LSD1 was performed using the Lance Ultra LSD1 Histone H3 Lysine 4 Demethylase Assay Kit (PerkinElmer). Tris-buffer (50 mMTris-HCl, 50 mMNaCl, 0.01% Tween-20, 1 mM DTT, 10 μM FAD, 10% glycerol, pH 9.0) was first prepared, and after preparation, the LSD1 fractions required for test were placed in this buffer and incubated for one hour at room temperature; the fractions included 4 μL of substrate solution (2.5 μL Bio-H3K4me2, 1-24aa), 4 μL of enzyme solution (4 nM LSD1), and 2 μL of inhibitor in a 384-well microplate. After these operations, another 5 μL of assay mixture containing Eu-labeled H3K4me0 antibody and ULight-Streptavidin anti-biotin protein was added to the system, after which the fluorescence intensity of the components to be tested was detected by using Envision (PerkinElmer) in TR-FRET mode (excitation wavelength of 320 nm, emission wavelength of 620 nm). The test was repeated a total of three times to minimize errors. The final IC50 data were calculated by the software GraphPad Prism 5, and finally the sigmoidal dose-response (variable slope) method was used to obtain the fitted curve. |
| Affinity data for this assay | |
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