| Assay Method Information | |
| | Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay |
| Description: | [Bcl-2 protein or Bcl-xL protein], [Probe], [Antibody],Protein Probe nM nM Antibody nMGST- F-Bak 1 100 Tb-anti- 1Bcl- Acetyl-(SEQ GST 2 ID NO: 3)-NH2The samples were then mixed on a shaker for 1 minute and equilibrated for an additional 3 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the Envision (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-GST antibody) emission filters. Inhibition constants (Ki) were determined using Wang's equation (Wang FEBS Lett. 1995, 360, 111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) to determine apparent half maximal inhibitory concentration (IC50) after HS protein binding. |
| Affinity data for this assay | |
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