Amplite® Fluorimetric Sphingomyelinase 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 | SDSProtocol |
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 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 fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 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 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 solution.
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/13621
Add 1 µL of 10 units/mL Sphingomyelinase standard solution into 1000 µL Assay Buffer (Component E) to generate a 10 mU/mL Sphingomyelinase standard solution. Take 10 mU/mL Sphingomyelinase standard solution and perform 1:2 serial dilutions to get serially diluted Sphingomyelinase standards (SMase7 - SMase1). Note: Diluted Sphingomyelinase standard solution is unstable. Use within 4 hours.
PREPARATION OF WORKING SOLUTION
1. Sphingomyelin working solution:
Add 50 µL of Sphingomyelin (Component B) into 5 mL of SMase Reaction Buffer (Component D) and mix well to make Sphingomyelin working solution. 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 them well. Then, add 25 µL of 200X Amplite™ Red stock solution into the bottle of Enzyme Mix solution to make Sphingomyelinase working 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.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of Sphingomyelinase standards and test samples in a solid black 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 Sphingomyelin 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 Sphingomyelinase assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Sphingomyelinase 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 fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
Images
Citations
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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