Invitrogen™ Amplex™ Red Sphingomyelinase Assay Kit

Invitrogen™ Amplex™ Red Sphingomyelinase Assay Kit

The Amplex™ Red Sphingomyelinase Assay Kit provides a sensitive, rapid, and simple fluorometric method for detecting very low concentrations of sphingomyelinase using a fluorescence microplate reader or fluorometer.

In this enzyme-coupled assay, sphingomyelinase activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex™ Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. Sphingomyelinase hydrolyses the sphingomyelin to yield ceramide and phosphorylcholine. Alkaline phosphatase is added, which hydrolyzes phosphorylcholine to form choline. The choline is then oxidized by choline oxidase to form betaine and hydrogen peroxide. In the presence of horseradish peroxidase, the hydrogen peroxide reacts with Amplex™ Red reagent in a 1:1 stoichiometric ratio to generate the highly fluorescent product resorufin.

Features

• Detects sphingomyelinase activity levels as low as 80 µU/mL in a 200 µL assay volume
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference