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Assay Method Information

Assay Name:  In Vitro hLTC4 Synthase Enzyme Assay
Description:  In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 methyl ester is converted to LTC4 methyl ester.In order to obtain IC50-values for the compounds, the following procedure was used:10 mL of 0.6 nM of human recombinant purified LTC4 synthase expressed from Pichia Pastoris in buffer 50 mM tris (pH 7.5), 0.05% BSA 0.03% DD M 100 mM NaCl was preincubated for 30 min with 10 mL of substrate mix containing 6 mM LTA4-methyl ester substrate and 1 mM glutathione in 50 mM Tris pH7.5 0.05% BSA 0.03% DDM 100 mM NaCl together with 0.1 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10−9.5-10−5 M as well as with DMSO only.The enzymatic reaction is initiated by addition of 10 μL LTC4S enzyme solution to 10 μL substrate solution to the assay plate containing 0.5 mL of compound in DMSO or 0.5 mL DMSO control-The reaction is stopped after 30 min by addition of 40 μL 75% (w/v) acetonitrile in H2O.After stopping the reaction the LTC4 methyl ester product was detected by LCMSMS,The detected product concentration, with background subtracted, is plotted versus compound concentration and IC50 is determined as 50% of maximum inhibition.
Affinity data for this assay
 

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Last update November 1, 2007
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