Thermo Fisher Scientific 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.

• 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

Assay Principle

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 hydrolyzes sphingomyelin to yield ceramide and phosphorylcholine. Alkaline phosphatase hydrolyzes phosphorylcholine to form choline, which is oxidized by choline oxidase to form betaine and hydrogen peroxide.
In the presence of horseradish peroxidase (HRP), hydrogen peroxide reacts with Amplex™ Red reagent in a 1:1 stoichiometric ratio to generate the highly fluorescent product resorufin.

Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, resulting in minimal interference from autofluorescence in most biological samples.

Applications

Use Amplex™ Red Assays for a broad range of investigations, including:

• Cell signaling and lipid studies
• Neurobiology research
• Inflammation and immune function
• Metabolism analysis

We also offer:

• Amplex™ UltraRed Reagent (Cat. No. A36006) – second-generation reagent with greater sensitivity and brighter fluorescence
• Amplex™ Red/UltraRed Stop Reagent (Cat. No. A33855) – allows termination of fluorescence signal generation at a user-determined time point; signal remains stable for at least three hours

Custom assay design and packaging options are available.

See our complete line of Fluorescence Microplate assays

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