| Assay Method Information | |
| | Assay for Determination of Inhibition of Rat Intestinal Alpha-Glucosidases |
| Description: | Various concentrations of test compounds were prepared in DMSO and then diluted into buffer consisting of 100 mM citric acid, 200 mM disodium phosphate, pH 6.0. Rat intestinal acetone powder (Sigma) was dissolved to 12.5 mg/mL in assay buffer and sonicated in an ice bath, centrifuged, and the supernatant was collected, aliquoted and stored at −20° C. until use. On the day of the assay, the rat intestinal lysate was diluted to 6.25 mg/mL (sucrose reactions) and 2.1 mg/ml (maltose reactions) in assay buffer. The final reaction solutions for sucrose contained 2.1 mg/mL rat intestinal lysate, 40 mM sucrose, 50 or 25 μM of test compound, and 3.3% DMSO. The final reaction solution for maltose contained 0.69 mg/mL rat intestinal lysate, 10 mM maltose, 50 or 25 μM of test compound, and 3.3% DMSO. The reaction was initiated by the addition of substrate (sucrose or maltose) and allowed to proceed at 37° C. for 20 min to assess rat intestinal alpha-glycosidase activities. The reaction was stopped by addition of 20 μL of 2 M Tris-HCl, pH 7.0. The glucose produced during the reaction was detected using a commercial Amplex Red glucose detection kit from Thermo Fisher. For a full plate, the Amplex Red detection mixture was prepared by mixing 35 μL glucose oxidase, 35 μL horseradish peroxidase and 35 μL Amplex Red into 3395 μL 1× Amplex Red detection buffer. A total of 30 μL of detection mixture was added to each well. |
| Affinity data for this assay | |
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