Assay Method Information

Assay Name:  IL4I1 Enzymatic Assay
Description:  Using this assay, the potency (EC50) of each compound was determined from a ten-point (1:3 serial dilution) 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 consisted 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 were in excess such that the conversion of H2O2 to resorufin product occurred
Affinity data for this assay
 

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