| Assay Method Information | |
| | LSD1 Inhibitory Activity Assay |
| Description: | Experimental steps and a method: In this experiment, the in vitro inhibitory activity of the compounds of the present disclosure against LSD1 on an enzyme level was tested by using an AlphaScreen method. The experimental steps are described as follows:a) preparation of a 1× buffer:preparing a 1× buffer (improved trihydroxymethylaminomethane buffer);b) continuous dilution of the compounds:transferring the compounds of the present disclosure to a porous plate by using Echo at a final DMSO concentration of 1%;c) preparation of an enzyme solution:preparing an enzyme solution in the 1× buffer;d) preparation of a substrate solution:adding polypeptide to the 1× buffer to prepare a substrate solution;e) transferring 5 μL of the enzyme solution or the 1× buffer to the porous plate;f) conducting incubation at room temperature for 15 min;g) adding 5 μL of the substrate solution to each well to induce a reaction;h) conducting incubation at room temperature for 40 min;i) preparing a 1× Alphalisa buffer;j) preparation of a 1× Alphalisa buffer solution of receptors and donors:adding 15 μL of a receptor and donor solution in dark light environment, and conducting incubation at room temperature for 60 min;k) reading an endpoint in an EnSpire Alpha mode; andl) data processing:calculating an inhibitory value according to an equation 1:Inh %=(Max−Signal)/(Max−Min)*100; and equation 1:calculating an IC50 value by using XL-Fit according to an equation 2:Y=Bottom+(Top-Bottom)/(1+(IC50/X)*HillSlope), equation 2:where Y indicates the inhibitory rate, and X indicates the concentration of a compound. |
| Affinity data for this assay | |
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