| Assay Method Information | |
| | Chitin Synthase Assays |
| Description: | Assays were performed in 96-wells microtiter plate with flat bottom. To each well of the plate, 100 無 of WGA solution was added at 50 痢/mL. After incubation at room temperature for at least 16 h the WGA solution was removed by triple washing with distill water. Wells were then blocked by adding 300 無 of BSA blocking buffer (20 mg/mL, 50 mM Tris-HCl, pH 7.5) and incubated for 3 h at room temperature. Microtiter plates were stored until use at -20 蚓 in blocking buffer. Before usage plates were thawed at 25 蚓 and thoroughly emptied. The standard reaction mixture in total volume 100 無 contains: UDP-GlcNAc (2.0 mM), GlcNAc (40 mM), MgCl2 (5.0 mM) and digitonin (0.1% w/v) dissolved in pH 7.5 Tris-HCl buffer (50 mM) containing test compounds at a range of concentrations from 0 mM (control) to 3.0 mM. The enzymatic reactions were started by the addition of 0.2 mg/mL solution of trypsin (1-10 無) and membrane preparation (5-20 無). Usually four different concentrations of trypsin were te |
| Affinity data for this assay | |
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