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| | In-vitro IDO1 Enzyme (Indoleamine 2,3-dioxygenase) Assay |
| Description: | Human indoleamine 2,3-dioxygenasel (hIDO1) catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N-formylkynurenine which can be converted to kynurenine (KYN) by deformylation. hIDO1 with an N-terminal His tag, expressed and purified from E. coli cells was procured from either Enzo LifeScienccs, NY, USA). Unless otherwise stated, all materials were procured from Sigma Aldrich, Mo., USA.The assay monitoring the conversion of L-tryptophan to KYN was carried out as follows. hIDO1 (10 nM) was incubated with tryptophan (30 μM) in the presence of ascorbic acid (20 mM), methylene blue (10 μM) and catalase (100 μg/mL) in potassium phosphate buffer (50 mM; pH 6.5) at 37C for 30 min. The reaction was terminated with 30% trichloroacetic acid (TCA) and further incubated at 65C for 15 min to fully convert N-formylkynurenine to KYN. The reaction mixture was then centrifuged to remove sediments and the KYN in the supernatant was estimated by UV-visible absorption spectroscopy at 360 nm using a Waters HPLC system fitted with a C-18 column or by LC/MS/MS. See, Sono, 1980, J. Biol. Chem., 255:1339-1345, which is herein incorporated by reference.Percent inhibition at each concentration of test compounds was determined by estimating the decrease in KYN with reference to the reaction control with 1% DMSO vehicle. Data were analyzed using nonlinear regression to generate IC50 values using Graph Pad Prism 5. |
| Affinity data for this assay | |
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