| Assay Method Information | |
| | IC50 Assay |
| Description: | Inhibitors were made in TRIS buffer (50mM Tris pH 7.4 containing 1% Triton X-100). Working solutions of purified GCPII (50ug/mL) were diluted in TRIS buffer to provide from 15% to 20% conversion of substrate to product in the absence of inhibitor. The incubation mixture (final volume 250μL) contained either 25μL of an inhibitor solution or 25μL TRIS buffer to 175μ TRIS buffer in a test tube, followed by the addition of PABGgG (25μL, 100mM). The enzymatic reaction was initiated by the addition of 25μL of the GCPII working solution and allowed to proceed for 15min with constant shaking at 37°C. The reaction was quenched by the addition of 25μL methanolic TFA (2% trifluoroacetic acid by volume in methanol) followed by vortexing. The quenched mixture was immediately buffered by the addition of 25μL K2HPO4 (0.1M), vortexed, and centrifuged. |
| Affinity data for this assay | |
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