| Assay Method Information | |
| | Glucokinase Activity Assay |
| Description: | Recombinant human liver glucokinase was expressed as a FLAG fusion protein in E. coli, and purified on ANTIFLAG M2 AFFINITY GEL (Sigma). The assay was carried out at 30° C. in a 96-well plate. In the plate was distributed 69 μl each of assay buffer (25 mM Hepes Buffer: pH=7.2, 2 mM MgCl2, 1 mM ATP, 0.5 mM TNAD, 1 mM dithiothreitol), to which was added 1 μl of a DMSO solution of the compound or DMSO as control. Then, 20 μl of pre-ice-cooled enzyme mixture (FLAG-GK, 20 U/ml G6PDH) was distributed thereto, to which was added 10 μl of 25 mM glucose as substrate to initiate the reaction (final glucose concentration=2.5 mM). After starting the reaction, the absorbance at 405 nm was measured every 30 seconds for 10 minutes to evaluate the compound based on the initial increase for 5 minutes. FLAG-GK was added so that the increase of absorbance after 5 minutes fell between 0.05 to 0.1 in the presence of 1% DMSO. |
| Affinity data for this assay | |
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