Introduction
The Mes-Red Phospholipase D Assay Kit provides a sensitive method for measuring phospholipase D (PLD) activity in vitro using a fluorescence microplate reader or fluorometer. In this enzyme-coupled assay, PLD activity is monitored indirectly using Mes-Red reagent, a sensitive fluorogenic probe for H2O2.1,2 First, PLD cleaves the phosphatidylcholine (lecithin) substrate to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H2O2. Finally, H2O2, in the presence of horseradish peroxidase, reacts with Mes-Red reagent in a 1:1 stoichiometry to generate the highly fluorescent product, resorufin. Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples. The kit can be used to continuously assay PLD enzymes with near-neutral pH optima, whereas PLD enzymes with acidic pH optima can be assayed in a simple twostep procedure. Experiments with purified PLD from Streptomyces chromofuscus indicate that the Mes-Red PLD Assay Kit.

Storage conditions

The kit is shipped on ice. Store all components at -20°C. Shelf life: 6 months after receipt.

For Research Use Only. Not For Use In Diagnostic Procedures.