| Assay Method Information | |
| | LSD1 Enzyme Activity Assay |
| Description: | The effects of compounds on LSD1 enzyme activity are detected by an HTRF technology to evaluate their inhibition levels on LSD1 enzyme activity. First, 90PM test compound mother solution (dissolved in DMSO) is diluted for a 5-fold concentration gradient with DMSO successively to obtain a total of 8 concentrations of compound working solution 1 (90×). Then, 8 concentrations of working solution 1 are diluted for a 30-fold concentration gradient, i.e, 2 μL of working solution 1 is sucked and added to 58 μL of Buffer, and fully shaken and mixed well on a vortex mixer to obtain a total of 8 concentrations of screening compound working solution 2 (3×). In a 384-well shallow white plate, 2 μL of 3×LSD1 (Activemotif, 31426) enzyme solution and 2 μL of compound working solution 2 (3×) are sequentially added to each well, mixed well, and incubated at room temperature for 15 min; 2 μL of 3×H3K4mel (Anaspec, AS-64355-025) substrate solution is sequentially added to each well, mixed well, and incubated at room temperature for 60 min; 2 μL of stop solution (containing 5.4 mM 2-PCPA) is sequentially added to each well, mixed well, and incubated for 15 min at room temperature; and 4 μL of Eu-anti H3K4 (PerkinElmer, TRF0404-D) and allophycocyanin (Prozyme, PJ27S) premixed antibody (1:1) solution is sequentially added to each well, mixed well, and incubated at room temperature for 60 min. The 384-well plate is placed on a multifunctional microplate reader for reading values, an excitation light wavelength is set to 337 nm, and values at 620 nm and 665 nm are recorded. |
| Affinity data for this assay | |
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