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| | Glucan Synthase Assay |
| Description: | The glucan synthase assay follows the incorporation of tritiated UDP-glucose into acid insoluble β-glucan as catalysed by the glucan synthase activity present in membrane preparations from Candida albicans and Aspergillus fumigatus. UDP-[3H]-glucose (ca 0.01 μCi) is added to 100 μl assay buffer (50 mM Tris-Cl, pH8.0, 8% glycerol, 1 mM EDTA, 1.5 mM KF, 1 mM DTT, 20 μM GTPγS, 600 μM UDP-glucose) and the reaction initiated by the addition of 2.5 μl of enzyme preparation. After incubation at 30°C. for 120 minutes, 10 μl of 30 mg/ml BSA is added with mixing and the reaction is terminated with 110 μl ice-cold 20% TCA. The precipitate is collected on a GF/B filter plate and washed three times with 200 μl water. Once dry, 200 μl of Microscint20 is added to each well and the plate is read on a Top-count scintillation counter. |
| Affinity data for this assay | |
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