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| | Enzyme Assay |
| Description: | For characterization of the enzymatic activity of endothelial lipase and the effect of inhibitors, the phospholipase-specific substrate 1,2-bis(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (manufacturer: Molecular Probes) was used. Hydrolysis of the A1 ester bond of this phospholipid by the enzyme releases the fluorescent dye Bodipy, which can be detected by measuring the fluorescence after separation by thin-layer chromatography on an HPTLC plate (silica gel 60, Merck) or directly in the reaction vessel. To prepare the substrate solution, 100 ug of 1,2-bis(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (manufacturer: Molecular Probes) were dissolved in 100 ul of DMSO and taken up in 2.4 mg of tripalmitin (Sigma) in 393 ul of chloroform which contained 20 mg/ml DOPcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine). |
| Affinity data for this assay | |
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