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Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit
Red Fluorescence
Sphingomyelinase (SMase) is an enzyme that is responsible for cleaving sphingomyelin (SM) to phosphocholine and ceramide. Activation of SMases plays an important role in the cellular response such as regulation of cell growth, cell differentiation, cell cycle arrest and programmed cell death. Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action, including lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase and alkaline SMase. Among them, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered to be the major factors for the production of ceramide in cellular stress responses. Our Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit provides one of the most sensitive methods for detecting acidic SMase activity or screening its inhibitors. The kit uses Amplite® Red as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). It can be used for measuring the SMase activity in blood, cell extracts or other solutions. The fluorescence intensity of Amplite® Red is proportional to the formation of phosphocholine, therefore to the SMase activity. The kit is an optimized "mix and read" assay compatible with HTS liquid handling instruments.
Sphingomyelinase (from human placenta) dose response was measured on a 96-well half-area black plate with Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit (13622) using a fluorescence microplate reader. 20 µL of SMase standard or control was incubated with 20 µL of sphingomyelin working solution at  37 °C for 3 hours, and then 20 µL of sphingomyelinase assay mixture was added into each well. The signals shown in the figure are the readings at Ex/Em = 540/590 nm (cut off at 570 nm) after 2 hours incubation at room temperature.
Sphingomyelinase (from human placenta) dose response was measured on a 96-well half-area black plate with Amplite® Fluorimetric Acidic Sphingomyelinase Assay Kit (13622) using a fluorescence microplate reader. 20 µL of SMase standard or control was incubated with 20 µL of sphingomyelin working solution at  37 °C for 3 hours, and then 20 µL of sphingomyelinase assay mixture was added into each well. The signals shown in the figure are the readings at Ex/Em = 540/590 nm (cut off at 570 nm) after 2 hours incubation at room temperature.
protocol
Protocol
sds
SDS
COA
CatalogSize
Price
Quantity
13622200 Tests
Price
 
Spectral properties
Excitation (nm)571
Emission (nm)584
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
Instrument settings
Fluorescence microplate reader
Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black
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Page updated on September 25, 2025