| Assay Method Information | |
| | In Vitro ERAP Activity Assay |
| Description: | The enzymatic activity of ERAP1 or 2 was assayed using L-AMC (L-Leucine-7-amido-4-methylcoumarin hydrochloride) or R-AMC (L-Arginine-7-amido-4-methylcoumarin hydrochloride) respectively. Hepes at 50 mM with 100 mM NaCl at pH 7 was used as buffer. Briefly, 60 nL of test compounds were added in 384-wells plates (dark, non-binding surface) by acoustic dispensing with nanoacoustic dispenser Echo (Labcyte) and pre-incubated 30 minutes at ambient temperature with 10 uL of ERAP 0.8 ug/mL or 1 ug/mL or vehicle. The reaction was then started with the addition of 10 uL of substrate at 10 uM. The final concentration of ERAP, substrate and DMSO was 0.5 ug/mL, 5 uM and 0.4% respectively. For the kinetic readout a Victor 3V (Perkin-Elmer) was used with excitation at 380 nm and emission at 450 nm. The fluorescence was measured each 3 minutes during one hour. The Z and Z' factors were calculated according to J.-H. Zhang, T. D. Y. Chung, K. R. Oldenburg, A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, J. Biomol. Screen., 4 (1999) 67-73. Data analysis was performed using Xlfit v 5.0 or GraphPad Prism v 4.0. |
| Affinity data for this assay | |
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