| Assay Method Information | |
| | Biological Assay |
| Description: | To identify optimal conditions for identifying novel inhibitors of CNDP2, enzyme assays were performed with varied sources of purified enzyme, concentrations of enzyme, peptide substrates, concentrations of peptide substrates and metal cofactors. Empirically-determined optimal conditions included 50 nM purified CNDP2 (Sino Biological) in Assay Buffer that contained 100 mM Tris pH 7.4, 100 mM NaCl, 100 microM MnCl2, 1 mM DTT, 1% DMSO and Met-His peptide substrate added at 1 mM (near the enzyme Km for this substrate). The reaction mixture was incubated for 30 minutes at 37° C. before it was quenched with an equal volume of 1% TCA. Free histidine was quantified after derivatization with a fluorophore (o-phthaldialdehyde from Sigma-Aldrich, excitation 340 nm, emission 460 nm). The reaction was conducted in an automation-friendly format to meet future screening throughput demands. A histidine titration standard curve was also included to enable conversion of fluorescence signal to molar concentration of histidine released by the hydrolysis reaction and to ensure the linearity of the fluorescence signal vs. free histidine. |
| Affinity data for this assay | |
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