| Assay Method Information | |
| | Method B: Inhibition of Mcl-1 by the Fluorescence Quenching Assay |
| Description: | The addition of a compound which binds competitively to the same site as the peptide will result in an increase in the fluorescence intensity of the protein due to displacement of the fluorescence quencher. An 11-point serial dilution of each compound was prepared in DMSO, the final buffer conditions were 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 150 mM NaCl, 0.05% Tween 20, pH 7.4 and 5% DMSO. The final protein concentration in the assay was 1 nM with the peptide present at 10, 20 or 400 nM. The experiments were incubated for 2 hours at room temperature before fluorescence intensity was measured on a Biotek SynergyNeo plate reader (Excitation 620 nm, emission 680 nm). The dose response curves were plotted with XL-Fit software using a 4-Parameter Logistic Model (Sigmoidal DoseResponse Model) and the inhibitory concentrations that gave a 50% increase in fluorescence intensity was determined (IC50). The Ki values were determined from the IC50 values according to Cer et al, Nucleic Acids Res, 2009, 37 (WebServer issue): W441-W445. Inhibition constants (Ki) are determined from complete binding inhibition curves (cKi) or from estimated from incomplete binding inhibition curves (eKi) in most cases due to low activity. |
| Affinity data for this assay | |
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