| Assay Method Information | |
| | UDP-Glo Glucosylceramide Synthase Biochemical Assay |
| Description: | Using Promega s UDP-Glo Glycosyltransferase assay kit (Promega Corporation, Madison, WI, USA (Promega)), GCS activity was indirectly measured by detecting the amount of UDP produced. An aliquot of GCS enzyme (1.5 µg crude golgi preparation, total protein) and titrated test compound were aliquoted to each well and incubated for 30 minutes at room temperature. Substrate mixture was prepared by mixing C6 ceramide (Avanti Polar Lipids, Alabaster, AL USA (Avanti)) (micelles prepared at 0.6 mM in 0.6 mM DOPC) and UDP-glucose (20 µM; Promega), at concentrations equivalent to 2x Km, in assay buffer (25 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2). An equivalent volume of substrate mixture was then added to each well. Following a 20 h incubation at room temperature to allow for GCS turnover of substrate, an equal volume of UDP detection reagent (Promega) was added to each well and incubated for an additional 75 minutes at room temperature to simultaneously convert the accumulated UDP product into ATP and generate light in a luciferase reaction. The generated light was detected using a luminometer. Random luminescence values (RLUs) were normalized to mean min and max effects, as determined on each plate. Min was defined as the mean of the values of the wells treated with vehicle (DMSO) and which represent 0% inhibition; max was defined as the mean of the values of the wells treated with a reference inhibitor and which represent the 100% effect. Values for %Emax and EC50 were determined by best-fitting the normalized data to a curve in Activity Base along a four-parameter logistic nonlinear regression (4PL) model (based on the Levenberg-Marquardt algorithm and defined by the equation below):where: n is 4PMin (bottom of the curve); m is 4PMax (top of the curve); i is IP (inflection point of curve); and p is slope. See Levenberg, K., A Method for the Solution of Certain Problems in Least Squares , Quart. Appl. Math.2, (1944), pp 164 168 and Marquardt, D., An Algorithm for Least Squares Estimation on Nonlinear Parameters , SIAM J. Appl. Math.11, (1963) pp 431 441. |
| Affinity data for this assay | |
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