Amplite® Colorimetric Sphingomyelinase Assay Kit *Blue Color*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
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Shipping | Standard overnight for United States, inquire for international |
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Platform
Absorbance microplate reader
Absorbance | 655 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare sphingomyelin working solution (50 µL)
- Add SMase standards and/or SMase test samples (50 µL)
- Incubate at 37°C for 1 - 2 hours
- Add sphingomyelinase working solution (50 µL)
- Incubate at RT for 1 - 2 hours
- Monitor absorbance at 655 nm
Important notes
Thaw one vial (or bottle) of each kit component at room temperature before starting your experiment.
PREPARATION OF STOCK SOLUTION
1. Sphingomyelinase standard stock solution (10 U/mL):
Add 20 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase Standard (Component F) to make a 10 units/mL sphingomyelinase standard stock solution.
2. Amplite™ UltraBlue stock solution (200X):
Add 100 µL of DMSO (Component G) into the vial of Amplite™ UltraBlue (Component C) to make 200X Amplite™ UltraBlue stock solution. Note: The Amplite™ UltraBlue is unstable in the presence of thiols (such as DTT and 2-mercaptoethanol). The final concentration of DTT or 2-mercaptoethanol in the reaction should be lower than 10 µM. Amplite™ UltraBlue is also unstable at high pH (>8.5). The reactions should be performed at pH 7 - 8. pH 7.4 is recommended for the assay buffer.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13620
Add 1 µL of 10 units/mL sphingomyelinase standard stock solution into 1000 µL assay buffer (Component E) to generate a 10 mU/mL sphingomyelinase standard. Take 500 µL of 10 mU/mL sphingomyelinase standard to perform 1:2 serial dilutions to get serially diluted sphingomyelinase standards (SMase7 - SMase1). Note: Diluted sphingomyelinase standard stock solution is unstable and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
1. Sphingomyelin working solution:
Add 50 µL of Sphingomyelin (Component B) into 5 mL SMase Reaction Buffer (Component D) and mix well. Note: The sphingomyelin working solution should be used promptly.
2. Sphingomyelinase working solution:
Add 5 mL of Assay Buffer (Component E) into the bottle of Enzyme Mix (Component A) and mix well. Add 50 μL of 200X Amplite™ UltraBlue stock solution into the bottle of Enzyme Mix solution before starting the assay. Note: The sphingomyelinase working solution should be used promptly and kept from light; longer storage is likely to cause high assay background. The cloudiness of the mixture is normal; it will not interfere with the assay performance.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of sphingomyelinase standards and test samples in a white wall/clear bottom 96-well microplate. SMase = Sphingomyelinase Standards (SMase1 - SMase7, 0.078 to 5 mU/mL), BL = Blank Control, TS = Test Samples.
BL | BL | TS | TS |
SMase1 | SMase1 | ... | ... |
SMase2 | SMase2 | ... | ... |
SMase3 | SMase3 | ||
SMase4 | SMase4 | ||
SMase5 | SMase5 | ||
SMase6 | SMase6 | ||
SMase7 | SMase7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
SMase1 - SMase7 | 50 µL | Serial Dilutions (0.078 to 5 mU/mL) |
BL | 50 µL | Assay Buffer |
TS | 50 µL | Test Sample |
- Prepare sphingomyelinase standards (SMase), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Treat your cells or tissue samples as desired.
- Add 50 µL of sphingomyelin working solution to each well of sphingomyelinase standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of sphingomyelin working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction mixture at 37°C for 1 - 2 hours.
- Add 50 µL of sphingomyelinase working solution to each well of sphingomyelinase standard, blank control, and test samples to make the total assay volume of 150 µL/well. For a 384-well plate, add 25 µL of sphingomyelinase assay working solution into each well instead, for a total volume of 75 µL/well.
- Incubate the reaction mixture for 1 - 2 hours at room temperature (protected from light).
- Monitor the absorbance increase with an absorbance microplate reader at 655 nm.
Images
Citations
Authors: Mashhadi Akbar Boojar, Masoud
Journal: Nova Biologica Reperta (2019): 275--283
Authors: Su, Yu-Ting and Meng, Xing-Xing and Zhang, Xi and Guo, Yi-Bin and Zhang, Hai-Jun and Cheng, Yao-Ping and Xie, Xiao-Ping and Chang, Yao-Ming and Bao, Jun-Xiang
Journal: The Anatomical Record (2017)
Authors: Li, Lin and Niu, Huanmin and Sun, Bin and Xiao, Yanan and Li, Wei and Yuan, Huiqing and Lou, Hongxiang
Journal: Toxicology and Applied Pharmacology (2016): 175--184
Authors: Mashhadi Akbar Boojar, Mahdi and Ejtemaei Mehr, Shahram and Hassanipour, Mahsa and Mashhadi Akbar Boojar, Masoud and Dehpour, Ahmad Reza
Journal: Iranian Journal of Pharmaceutical Research (2016): 421--433
Authors: Baixauli, Francesc and Acín-Pérez, Rebeca and Villarroya-Beltrí, Carolina and Mazzeo, Carla and Nunez-Andrade, Norman and Gab, undefined and é-Rodriguez, Enrique and Ledesma, Maria Dolores and Blázquez, Alberto and Martin, Miguel Angel and Falcón-Pérez, Juan Manuel and others, undefined
Journal: Cell metabolism (2015): 485--498
Authors: Davis, Warren
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2014): 168--179
Authors: Xu, Miao and Liu, Ke and Southall, Noel and Marugan, Juan J and Remaley, Alan T and Zheng, Wei
Journal: Analytical and bioanalytical chemistry (2012): 407--414
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