| Assay Method Information | |
| | IL4I1 Enzymatic Assay |
| Description: | nterleukin 4 inducible protein 1 (IL4I1) is an L-amino oxidase that catalyzes the oxidation of aromatic residues (Phe, Trp and Tyr): L-amino acid+H2O+O2→2-oxo acid+NH3+H2O2. Equal molar of H2O2 and the corresponding alpha-ketoacid are produced when IL4I1 and substrate are added. In this assay, the hydrogen peroxide generated by IL4I1 are then detected through a coupled reaction with Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and Horse Peroxidase (HRP) to produce Resorufin product that could be detected in the form of fluorescence signals. The assessment of the inhibitory effect of small molecules (EC50) on IL4I1 is measured by the effectiveness of the compounds to inhibit the production of H2O2.Using this assay, the potency (EC50) of each compound was determined from a ten-point (1:3 serial dilution; top compound concentration of 10000 nM) titration curve using the following outlined procedure. To each well of a black flat-bottom Greiner (Cat #781076) 384 well-plate, 25 nL of compound (0.1% DMSO in final assay volume of 25 μL) was dispensed, followed by the addition of 12.5 μL of 1× assay buffer (50 mM Hepes 7.0 and 0.005% Tween20 (Sigma, Cat #P8341; low peroxide grade)) containing 2 nM of recombinant IL4I1 (R&D Systems, Cat #5684-AO-020). Plates were placed in an ambient temperature humidified chamber for a four-hour pre-incubation with compound. Subsequently, each reaction was initiated by the addition of 12.5 μL 1× assay buffer containing 2 mM of each aromatic amino acids (Phe/Tyr/Trp), 0.1 mM Amplex Red and 2 U/mL of HRP. The final reaction in each well of 25 μL consists of 1 nM of IL4I1, 1 mM of each residues (Phe, Tyr and Trp), 0.05 mM Amplex Red and 1 U/mL of HRP. It should be noted that the concentrations of Amplex Red and HRP used here are in excess such that the conversion of H2O2 to Resorufin product occurs instantaneously and non-rate limiting. |
| Affinity data for this assay | |
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