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Assay Method Information

Assay Name:  Inhibition Spectra Assay
Description:  Formation of kynurenic acid (KYNA) is indirectly assessed by a decrease in light absorbance at 370 nm (OD370) as the L-kynurenine (KYN) substrate is converted by the human KAT II (hKAT II) enzyme into KYNA. An inhibitor would therefore inhibit the decrease in OD370.The protocol was performed by placing the following reagents into a Costar 384 well black plate (30 uL total assay volume/well): 10 uL of 3x concentrated compound; 10 uL of 3x concentrated substrate mix (BGG (Sigma G-5009); 3 mM L-Kynurenine in 150 mM Tris Acetate (Sigma K3750); 3 mM alpha-ketoglutaric acid in 150 mM Tris Acetate (Sigma K2010); and 210 uM pyridoxal 5-phosphate (PLP) in 150 mM Tris Acetate (Sigma 9255)); and 10 uL of 3x concentrated enzyme (15 nM enzyme in 150 mM Tris Acetate with 0.3% bovine serum).Plates were sealed and incubated at 37 C. for 15-20 h before reading OD370 on a SpectraMax Plus plate reader.
Affinity data for this assay
 

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Last update November 1, 2007
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