Invitrogen™ Amplex™ Red Sphingomyelinase Assay Kit

• 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

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.

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.

Use Amplex™ Red Assays for a Broad Range of Investigations

A wide variety of validated Amplex™ Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex™ UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex™ Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex™ Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.