| Assay Method Information | |
| | Biological Activity Assay |
| Description: | Proteins: Bcl-2 (Cisbio 63ADK000CB01PEG); Bcl-xL (Cisbio 63ADK000CB04PEG);Experimental procedures: a) Dilution of the compounds in DMSO: the compounds to be tested (10 mM stock solutions) were diluted 500 times with DMSO as the first concentration, and the resulting solutions were diluted using a 3-fold serial dilution, resulting in a total of 10 concentration gradients, and the 11th concentration was DMSO control. b) 100 nL/well of the compounds (which were prepared in the step a) was added to a 384-reaction plate (6008280, PerkinElmer) using ECHO, and the plate was centrifugated at 1000 rpm for 1 minute for future use. c) 5 μL/well of BCL-2 solution (2 nM) and BCL-X1 (5 nM), respectively, was added to the 384-reaction plate added with compounds as described above, and the plate was centrifugated at 1000 rpm for 1 minute; to which 5 μL/well of BAK (80 nM) was added, and the plate was centrifugated at 1000 rpm for 1 minute and incubated at 25° C. for 15 minutes. The concentrations of the compounds were started from 100 nM as the initial concentration and diluted with a 3-fold serial dilution to obtain 10 plus 0 points in total. The final concentration of DMSO was 0.5%. d) 10 μL/well of Anti-Tag1-Eu3+& Anti-Tag2-XL665 solution (cisbio) was added to the 384-reaction plate described above, and the plate was centrifugated at 1000 rpm for 1 minute and incubated at 25° C. for 180 minutes. e) The ratio of 665/615 nm was read with a Envision Multi-Function Reader. The inhibition constant (Ki) of the compounds of the present disclosure is determined in this manner, wherein the Ki of the compounds is expressed as < (less than) a specific value, meaning that the binding affinity value is lower than the detection limit of the test used. Wang's Competitive Binding of Two Different Ligands to a Protein Molecule. |
| Affinity data for this assay | |
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