| Assay Method Information | |
| | BPGM Phosphatase Assay |
| Description: | The following materials were used in this assay: Tris buffer (50 mM Tris, 0.01% Tween 20, pH 7.4); cyclohexylammonium salt of 2,3-BPG (stock solution of 500 mM in ultrapure distilled water; human Bisphosphoglycerate mutase (hBPGM; stock solution of 110 μM in Standard Buffer); 3-Phosphoglyceric acid (3-PG; stock solution of 100 mM in ultrapure distilled water); 384-Well Polystyrene Plates (Fisherbrand , Catalog No.: 12-566-625); BIOMOL GREEN REAGENT (Enzo life sciences, BML-AK111-0250); and 1 mM DMSO stock of exemplary compounds. A 3-PG enzyme mix solution (final enzyme concentration 62.5 nM, 3-PG concentration at 50 M, 14.5 uL) was added to onto a 384-well clear plate. Solutions of the test compounds were then added to the plate and the DMSO content was normalized across the plate. The plate was incubated at RT for 15 min. Next, the substrate 2,3-BPG (5 μL) was added to all the wells, and the plate was sealed and incubated for 24 hr at RT. After incubation, BIOMOL green dye (40 μL) was added to all the wells, the plate was shaken to mix, and the plate was incubated at RT for 20-30 min. Absorbance measurements were recorded at 620 nm. |
| Affinity data for this assay | |
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