Assay Method Information | |
| Peptidyl Prolyl Isomerase (PPIase) Inibition Assay |
Description: | PPIase(Rotamase) activity of FKBP12 was assayed using the peptide N-succinyl Ala-Leu-Pro-Phe p-nitroanilide as substrate. It is based on the observation that chymotrypsin cleaves the C-terminal amide bond only in the trans conformer of the chromogenic substrate. The rapid hydrolysis perturbs the cis-trans conformational equilibrium, which allows one to monitor the PPIase-catalyzed cis-to-trans isomerization. The absorbance at 390 nM versus time was monitored for up to 400 s. Rate constants were generated from the absorbance versus time plots at 8-12 compound concentrations. The Kiapp was determined from the corrected rate constants using an IC50 equation or a tight-binding equation by Morrison. |
Affinity data for this assay | |
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