| Assay Method Information | |
| | Inhibition Assay (pH 8.0) |
| Description: | 10 mM compound stock solutions were prepared in DMSO. For IC50 determination compound stocks were serially diluted (1:3) in DMSO.All measurements were performed with an EnSpire Perkin Elmer multimode reader using glutaminyl-7-amino-4-methylcoumarin (H-Gin-AMC) as substrate and recombinant pyroglutamyl aminopeptidase (pGAP) as auxiliary enzyme. Reactions were carried out at ambient temperature in black 96-well half area microplates. Each sample consisted of 1 μl test compound solution or solvent (DMSO) and 49 μl QC appropriately diluted in assay buffer (50 mM Tris/HCl, pH 8.0 or 50 mM MES buffer, pH=6.0). After a 10 min preincubation at ambient temperature the enzyme reaction was started by adding 50 μl of Gln-AMC-substrate/pGAP mixture in assay buffer. Final substrate concentrations were 50 and 200 μM for measurement at pH 8.0 or 6.0, respectively. Release of flourogenic AMC were recorded at excitation/emission wavelengths of 380/460 nm. Initial velocity of the enzyme reaction was calculated by linear regression of the first 10 data points using the Enspire Manager software. Final evaluation and calculation of IC50s were performed using GraphPad Prism software. IC50 values were calculated from normalized data (QC activity without inhibitor=100%) by nonlinear regression according to a 4-parameter logistic equation.Ki-values were calculated according to the following formula: Ki=IC50/(1+[S]/Km), where:[S] reflects to the concentration of substrate in the assay (50 μM for pH 8.0) and Km is the respective Michaelis-Menten constant (62 μM at pH 8.0). |
| Affinity data for this assay | |
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