| Assay Method Information | |
| | Amplex Red Assay of PldA |
| Description: | In a 96-black well plate, in a final reaction volume of 200 μL, the following components were combined on ice to yield final concentrations of 50 mM Tris (pH 7.5), 80 mM KCl, 5 mM MgCl2, 5 mM CaCl2, 50 μM Amplex Red reagent, 0.1 U/mL choline oxidase, 1 U/mL horseradish peroxidase, 1.9 nM PldA, 20 μM (or 5 μg/mL) test compound, and 0.5 mM 7:0/7:0 PC. Lipid in chloroform was dried under a N2 stream in a glass test tube and resuspended in 50 mM Tris (pH 7.5) and 80 mM KCl buffer. All components were combined on ice, and the reaction was initiated byincubating at 37 °C and reading fluorescence emission at 590 nm (excitation = 530 nm) continuously at 90 s intervals for 60 min using a fluorescence plate reader. The signal was normalized by subtracting the background signal produced in the presence of DMSO and absence of PldA. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |