| Assay Method Information | |
| | In Vitro Screening Test |
| Description: | Compound solutions: The products (i.e., the compounds or the hydrochloride salt of compounds) obtained in the foregoing preparation examples of the present invention were each independently dissolved in DMSO to form solutions at suitable concentrations;Buffer I: 50 mmol/L HEPEs-Tris, pH=6.5, 5 mmol/L magnesium chloride, 10 mol/L valinomycin (J&K Scientific Ltd., goods No.: 227304);Buffer II: 50 mmol/L HEPEs-Tris, pH=6.5, 5 mmol/L magnesium chloride, 10 mol/L valinomycin, 5 mmol/L potassium chloride;5-adenosine triphosphate (ATP, Sigma-Aldrich, goods No.: A2383) solution: Dilute ATP with buffer I to 5 mM;Malachite green solution: Dissolve 0.12 wt % malachite green (J&K Scientific Ltd., goods No.: 913120) in 2.5 mol/L sulfuric acid, 7.5 wt % ammonium molybdate (J&K Scientific Ltd., goods No.: 128321) and 11% Tween 20(V/V). During use, sulfuric acid, ammonium molybdate and Tween 20 are mixed in a ratio of 100:25:2;Rabbit gastric mucosal microsomes (rich in H+/K+-ATPase, self-extracted), extracted by sucrose gradient centrifugation: The rabbit stomach was washed with tap water and 3M NaCl solution, respectively, and then the surface water was removed with filter paper. A pre-cooled homogenization buffer (4 ml/g tissue) was added and homogenized for 2-5 min in a tissue homogenizer. After the homogenizing, if there are large tissue particles, they can be removed by centrifugation (600 g, 10 min). The supernatant was then transferred to a clean centrifuge tube and centrifuged at 20,000 g for 30 min, then the supernatant was transferred to a clean centrifuge tube and further centrifuged at 100,000 g for 90 min and the precipitate was collected; the precipitate was suspended in homogenate and blown evenly, and the protein concentration was measured by the Bradford's method and adjusted to 10 mg/ml; 7.5 wt % Ficoll's layering solution was added in equal proportions. After centrifugation at 100,000 g for 60 min, the middle layer (H+/K+-ATPase-enriched gastric membranes) was collected in a clean centrifuge tube, diluted with homogenate by 4-5 times, and then centrifuged at 100,000 g for another 90 min, and the precipitate was collected; the precipitate was suspended in homogenate and homogenized in a glass homogenizer, and the protein concentration was measured by the Bradford's method and adjusted to 22.5 mg/ml. It was frozen at −80° C. and stored for future use. |
| Affinity data for this assay | |
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