Invitrogen™ Amplex™ Red Sphingomyelinase Assay Kit 

Description

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. Because resorufin has absorption and fluorescence emission maxima of approximately 571nm and 585nm, respectively, there is little interference from autofluorescence in most biological samples.

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

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, a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex Red/UltraRed Stop Reagent. 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.

Enzyme and Protein Activity Assays, Lipase and Phopholipase Activity, Proteins, Expression, Isolation and Analysis

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