Acidic Sphingomyelinase Assay Kit (Fluorimetric) (ab190554)
Key features and details
- Assay type: Semi-quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Sample type: Cell culture extracts, Tissue Extracts
- Sensitivity: 1 U/ml
Overview
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Product name
Acidic Sphingomyelinase Assay Kit (Fluorimetric)
See all Acid sphingomyelinase kits -
Detection method
Fluorescent -
Sample type
Cell culture extracts, Tissue Extracts -
Assay type
Semi-quantitative -
Sensitivity
1 U/ml -
Species reactivity
Reacts with: Other species
Predicted to work with: Mammals
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Product overview
Acidic Sphingomyelinase Assay Kit (Fluorometric) (ab190554) provides one of the most sensitive methods for detecting acidic sphingomyelinase (SMase) activity in cell extracts, or for screening the effect of inhibitors on acid SMase activity. The kit uses our AbRed Indicator as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). The fluorescence intensity of AbRed is proportional to the formation of phosphocholine, therefore proportional to the SMase activity.
This product can be used for measuring the SMase activity in cell extracts or solutions such as blood. The kit is an optimized “mix and read” assay which is compatible with HTS liquid handling instruments.
This assay is semi-quantitative as it does not contain a SMase standard for calibration. When a known concentration of sphingomyelinase is used, the assay can detect as low as 1 U/mL acidic sphingomyelinase in solution.
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Notes
Sphingomyelinase (SMase; sphingomyelin phosphodiesterase, EC 3.1.4.12) is responsible for cleaving sphingomyelin (SM) to phosphocholine and ceramide. Activation of SMase plays an important role in cellular responses such as regulation of cell growth, cell differentiation, cell cycle arrest and programmed cell death. Five types of sphingomyelinase have been identified, based on their cation dependence and optimal pH of action: lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase and alkaline SMase. Among those five types, lysosomal acidic SMase and magnesium-dependent neutral SMase are considered to be the major factors for the production of ceramide in cellular stress responses.
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Platform
Microplate reader
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 200 tests AbRed Indicator 1 vial Assay Buffer 1 x 10ml DMSO 1 x 200µl Enzyme Mix 2 vials SMase Reaction Buffer 1 x 10ml Sphingomyelin 1 x 100µl -
Research areas
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Function
Converts sphingomyelin to ceramide. Also has phospholipase C activities toward 1,2-diacylglycerolphosphocholine and 1,2-diacylglycerolphosphoglycerol. Isoform 2 and isoform 3 have lost catalytic activity. -
Involvement in disease
Defects in SMPD1 are the cause of Niemann-Pick disease type A (NPDA) [MIM:257200]; also known as Niemann-Pick disease classical infantile form. It is an early-onset lysosomal storage disorder caused by failure to hydrolyze sphingomyelin to ceramide. It results in the accumulation of sphingomyelin and other metabolically related lipids in reticuloendothelial and other cell types throughout the body, leading to cell death. Niemann-Pick disease type A is a primarily neurodegenerative disorder characterized by onset within the first year of life, mental retardation, digestive disorders, failure to thrive, major hepatosplenomegaly, and severe neurologic symptoms. The severe neurological disorders and pulmonary infections lead to an early death, often around the age of four. Clinical features are variable. A phenotypic continuum exists between type A (basic neurovisceral) and type B (purely visceral) forms of Niemann-Pick disease, and the intermediate types encompass a cluster of variants combining clinical features of both types A and B.
Defects in SMPD1 are the cause of Niemann-Pick disease type B (NPDB) [MIM:607616]; also known as Niemann-Pick disease visceral form. It is a late-onset lysosomal storage disorder caused by failure to hydrolyze sphingomyelin to ceramide. It results in the accumulation of sphingomyelin and other metabolically related lipids in reticuloendothelial and other cell types throughout the body, leading to cell death. Clinical signs involve only visceral organs. The most constant sign is hepatosplenomegaly which can be associated with pulmonary symptoms. Patients remain free of neurologic manifestations. However, a phenotypic continuum exists between type A (basic neurovisceral) and type B (purely visceral) forms of Niemann-Pick disease, and the intermediate types encompass a cluster of variants combining clinical features of both types A and B. In Niemann-Pick disease type B, onset of the first symptoms occurs in early childhood and patients can survive into adulthood. -
Sequence similarities
Belongs to the acid sphingomyelinase family.
Contains 1 saposin B-type domain. -
Cellular localization
Lysosome. - Information by UniProt
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Alternative names
- Acid sphingomyelinase
- ASM
- ASM_HUMAN
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Images
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Sphingomyelinase Dose Response
Sphingomyelinase (purified from human placenta) dose response was measured on a 96-well half-are black plate following assay procedure, using a Gemini fluorescence microplate reader (Molecular Devices). 20 µL of SMase solution 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. Signals shown in the figure correspond to the readings at Ex/Em = 540/590 nm (cut off at 570 nm) after 2 hours incubation at room temperature.
