| Assay Method Information | |
| | In Vitro Enzyme Inhibition Assay - LSD1 |
| Description: | The effect of the compounds on LSD1 enzyme activity was examined using the HTRF technique to assess the level of inhibition. Initially, a master solution of 90 μM test compound (dissolved in DMSO) was prepared and sequentially diluted with DMSO in a 5-fold concentration gradient to yield a total of 9 concentrations of the compound working solution 1 (90×). Subsequently these 9 concentrations of working solution 1 were sequentially diluted by a 30-fold concentration gradient, i.e., 2 μL of working solution 1 was aspirated and added to 58 μL of Buffer, mixed on a vortex mixer with sufficient shaking to yield 9 concentrations of the screening test compound working solution 2 (3×). In a 384-well shallow white plate, 2 μL of the compound working solution 2 (3×) was added to each well, followed by the addition of 2 μL of a 6×LSD1 (Activemotif, 31426) and 6×FAD (Sigma, F8384) pre-mixed (1:1) solution. The mixture was incubated at room temperature for 15 minutes. Then 2 μL of the 3×H3K4mel (Anaspec, AS-64355-025) substrate solution was added to each well, mixed evenly, and incubated for 60 minutes. Afterward, 2 μL of a termination solution (containing 5.4 mM 2-PCPA) was added to each well, mixed evenly, and incubated for another 15 minutes. Finally 4 μL of a pre-mixed antibody solution of Eu-anti H3K4 (PerkinElmer, TRF0404-D) and allophycocyanin (Prozyme, PJ27S) (1:1) was added to each well, mixed evenly, and incubated for 60 minutes. The 384-well plate was then read on a multifunctional microplate reader, with the excitation wavelength set to 337 nm. Readings were recorded at 620 nm and 665 nm. The data results were presented as the ratio of the 665 nm signal value to the 620 nm signal value in each well, calculated using the formula: Ratio=104×665 nm signal value/620 nm signal value. |
| Affinity data for this assay | |
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