| Assay Method Information | |
| | GO Biochemical Assay |
| Description: | GO biochemical enzymatic reaction was performed in black 384-well low binding plate in a total volume of 25 μL. The reaction mixture contained 5 nM GO, 100 μM glycolate, 0.1 U/mL HRP, 50 μM Amplex Red, and 1:3 serial diluted test compounds in a buffer containing 50 mM Tris pH 7.8, 0.0025% Tween-20, and 0.02% BSA. Twenty five nanoliter of 1000× test compounds were pre-spotted onto 384-well low binding plate by Echo 555 Liquid Handler (Labcyte Inc., San Jose, CA) with starting final concentration of 10 μM, followed by addition of 5 μL/well of 25 nM GO (5× of 5 nM final concentration) and incubated for 15 min. Ten microliter of 2.5× of 0.1 U/mL final concentration HRP was added to each well, followed by addition of 10 μL 2.5× 100 μM final concentration of glycolate substrate and of 2.5× 50 μM final concentration of Amplex Red. The reaction was mixed and incubated at room temperature for 20 min followed by reading the plates by EnVision plate reader (Perkin Elmer, San Jose, CA) with excitation at 570 nm and emission 585 nm. The wells with DMSO were used as negative controls (as 0% inhibition) whereas wells without GO enzymes were used as positive controls (as 100% inhibition). The % inhibition as calculated as 100%×(Well-Negative)/(Positive-Negative |
| Affinity data for this assay | |
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