| Solution Information | help | |
| Enzyme: | Mannose-6-phosphate isomerase | |
| inhibitor: | BDBM43816 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | PMI assay materials: 1) Human PMI protein was provided by Dr. Hudson Freeze (Sanford-Burnham Medical Research Institute, San Diego, CA). 2) Substrate working solution: 50 mM HEPES, pH 7.4, 2.0 mM Mannose-6-phosphate, 1.6 U/ml Diaphorase, 0.2 mM Resazurin. 3) Enzyme working solution: 50 mM HEPES, pH 7.4, 0.44 mM NADP+, 9.048 mM MgCl2, 0.01% Tween 20, 4.6 ug/ml phosphoglucose isomerase, 30 ng/ml PMI, 1.8 ug/ml G6PDH. PMI Dose-response confirmation protocol: 1) 9 uL of Substrate working solution was added to columns 3-24 of a Greiner 384-well black plate (cat # 784076) using a WellMate bulk dispenser (Matrix) 2) 9 ul of Substrate working solution without mannose-6-p was added to columns 1 and 2 (positive control) 3) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4) 2 uL compounds in 10% DMSO were transferred into columns 3 | |
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