Amplite® Fluorimetric Sphingomyelin Assay Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 571 |
Emission (nm) | 584 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare SMase working solution (50 µL)
- Add sphingomyelin standards or test samples (50 µL)
- Incubate at 37 °C for 1 - 2 hours
- Add sphingomyelin assay mixture (50 µL)
- Incubate at RT for 0.5 - 2 hours
- Monitor fluorescence intensity at Ex/Em = 540/590 nm (cut off at 570 nm)
Important notes
Thaw kit components at room temperature before starting your experiment.
PREPARATION OF STOCK SOLUTION
1. SMase stock solution (100X):
Add 50 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase (Component B) to make SMase stock solution (100X).
2. Amplite™ Red stock solution (200X):
Add 80 µL of DMSO (Component G) into the vial of Amplite™ Red (Component C) to make 200X Amplite™ Red stock solution. Keep from light. Note: The Amplite™ Red 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™ Red 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/13625
Add 2 µL of 50 mM Sphingomyelin (Component F) into 1000 µL of SMase Reaction Buffer (Component D) to get a 100 µM Sphingomyelin standard solution (SM7). Take 100 µM Sphingomyelin standard solution to perform 1:3 serial dilutions to get serially diluted sphingomyelin standards (SM6 - SM1).
PREPARATION OF WORKING SOLUTION
1. Sphingomyelinase (SMase) working solution:
Prepare SMase working solution by adding the whole content (50 µL) of 100X SMase stock solution into 5 mL of SMase Reaction Buffer (Component D) and mix well. Note: The SMase working solution should be used promptly.
2. Sphingomyelin working solution:
Add the whole content (5 mL) of Assay Buffer (Component E) into the bottle of Enzyme Mix (Component A) and mix well. Add 25 µL 200X Amplite™ Red stock solution into the bottle of Enzyme Mix solution to make the sphingomyelin assay mixture before starting the assay. Note: The sphingomyelin assay mixture should be used promptly and kept from light; longer storage is likely to cause high assay background.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of sphingomyelin standards and test samples in a solid black 96-well microplate. SM = Sphingomyelin Standards (SM1 - SM7, 0.1 to 100 µM), BL = Blank Control, TS = Test Samples.
BL | BL | TS | TS |
SM1 | SM1 | ... | ... |
SM2 | SM2 | ... | ... |
SM3 | SM3 | ||
SM4 | SM4 | ||
SM5 | SM5 | ||
SM6 | SM6 | ||
SM7 | SM7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
SM1 - SM7 | 50 µL | Serial Dilutions (0.1 to 100 µM) |
BL | 50 µL | SMase Reaction Buffer |
TS | 50 µL | test sample |
- Prepare sphingomyelin standards (SM), 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 SMase working solution to each well of sphingomyelin standard, blank control, and test samples to make the total sphingomyelin assay volume of 100 µL/well. For a 384-well plate, add 25 µL of SMase 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 sphingomyelin assay mixture to each well of sphingomyelin standard, blank control, and test samples to make the total sphingomyelin assay volume of 150 µL/well. For a 384-well plate, add 25 µL of sphingomyelin assay mixture 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 fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (cut off at 570 nm).
Images
Citations
Authors: Wang, Yang and Chen, Yan-Yan and Gao, Gui-Bin and Zheng, Yang-Han and Yu, Nan-Nan and Ouyang, Lan and Gao, Xuejuan and Li, Nan and Wen, Shi-Yuan and Huang, Shangjia and others,
Journal: Molecular Therapy (2023)
Authors: Prymas, Kamila and Swiatkowska, Anna and Traczyk, Gabriela and Ziemlińska, Ewelina and Dziewulska, Anna and Ciesielska, Anna and Kwiatkowska, Katarzyna
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2019): 158549
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
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Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2014): 168--179
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Application notes
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