| Assay Method Information | |
| | BPGM Synthase Assay |
| Description: | The following materials were used in this assay: deionized water; Bisphosphoglycerate mutase (BPGM; Standard Buffer: 50 mM Tris, 0.01% Tween 20, pH 7.4); BPGM Reaction Buffer (50 mM Tris, 0.01% Tween 20, 3.23 mM KH2PO4, pH 7.4); dithiothreitol (DTT; Stock solution: 100 mM in Standard Buffer); 384-Well Black Assay Plate (Corning, P/N 3575); DMSO; BPGM Stock Solution (500 nM in BPGM Standard Buffer); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Stock Solution: 40 U/mL in 20 mM Tris-HCL Buffer with 20% Glycerol, 1 mM EDTA, 1 mM DTT); Glyceraldehyde 3-phosphate (GAP; Stock Solution: 20 mM in BPGM Standard Buffer); 3-PG Stock Solution (500 μM in BPGM Standard Buffer); Nicotinamide adenine dinucleotide (NAD; Stock Solution: 50 mM in BPGM Standard Buffer); Koningic Acid (1 mM Stock in DMSO); Resazurin Stock Solution (10 mM in DMSO); Diaphorase Stock Solution (40 U/mL in BPGM Standard Buffer); DTT Reaction Buffer (1 mM DTT in BPGM Reaction Buffer); Enzyme Solution (5 nM BPGM, 0.4 U/mL GAPDH in DTT Reaction Buffer); GAPDH Solution (0.4 U/mL GAPDH in DTT Reaction Buffer); Substrate Solution (2 mM NAD, 20 μM 3PG, 800 μM GAP in DTT Reaction Buffer); and Resazurin Solution (5 U/mL Diaphorase, 500 μM Resazurin in BPGM Standard Buffer). To the assay plate was added Enzyme Solution (20 uL) to columns 2-22 and 24 of the assay plate. To columns 1 and 23 was added GAPDH Solution (20 uL). A separate compound plate was prepared at the desired concentrations and all wells were normalized with 5% DMSO. The compound plate was incubated for 30 minutes at RT. Next, the substrate solution (20 μL) was dispensed to all reaction wells and the plate was incubated for 60 minutes at RT. Koningic acid (1 μL) was added and the plate was incubated for 10 minutes at RT. During this time period, the NADH fluorescence was read at ex: 360/em: 460. Next, Resazurin Solution (10 μL) was added to all reaction wells and the plate was incubated for 30 minutes at RT. The resorufin fluorescence was then read at ex:544/em:590. |
| Affinity data for this assay | |
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