| Assay Method Information | |
| | Molecular Level Activity Test of Anti-Apoptotic Protein BCL2 |
| Description: | The detection of the binding ability between anti-apoptotic protein BCL2 and pro-apoptotic protein Bim was performed using Homogeneous Time-Resolved Fluorescence technology. The reaction of this method occurred in a white shallow 384-well plate, with a total reaction volume of 10 μL. Specifically, it included 2 μL of the compound to be tested (2% DMSO), 4 μL His-tagged recombinant protein, and 4 μL Biotin-tagged BIM protein peptide. After 1 hour of reaction, 5 μL of each Anti-His and streptavidin-tagged XL665 antibody diluents diluted with a detection buffer were added. After incubating at room temperature for 4 hours, the values were read using the Envision multifunctional microplate reader to detect the effect of the compound to be tested on the binding ability of BCL2 to Bim protein peptides. The Envision parameter settings were exciting light at 320 nm, and emitting light at 615 nm and 665 nm. The binding ability of anti-apoptotic proteins to Bim protein peptides was indirectly reflected by the signal ratio of 665 nm and 615 nm. In the reaction, background wells without BCL2 and active wells for full binding of recombinant proteins without compounds and Bim protein peptides were set up. |
| Affinity data for this assay | |
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