Amplite® Fluorimetric D-Lactate Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
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 D-Lactate working solution (50 µL)
- Add D-Lactate standards or test samples (50 µL)
- Incubate at room temperature for 30 min - 2 hours
- Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570 nm)
Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NAD stock solution (100X):
Add 100 µL of H2O into the vial of NAD (Component C) to make 100X NAD stock solution.
2. D-Lactate standard solution (100 mM):
Add 200 µL of H2O or 1x PBS buffer into the vial of D-Lactate Standard (Component D) to make 100 mM D-Lacate standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13810
Add 10 µL of 100 mM D-Lactate standard solution into 990 µL 1x PBS buffer to generate 1 mM D-Lactate standard solution (SD7). Take 1 mM D-Lactate standard solution (SD7) and perform 1:3 serial dilutions in 1x PBS buffer to get serially diluted D-Lactate standards (SD6 - SD1). Note: Diluted D-Lactate standard solution is unstable, and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Mix (Component A), and mix well.
2. Add 50 µL of 100X NAD stock solution into the bottle of Component A+B, and mix well to make D-Lactate working solution. Note: This D-Lactate working solution is enough for one 96-well plate. Protect from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of D-Lactate standards and test samples in a solid black 96-well microplate. SD= D-Lactate Standards (SD1 - SD7, 1 to 1000 µM), BL=Blank Control, TS=Test Samples.
BL | BL | TS | TS |
SD1 | SD1 | ... | ... |
SD2 | SD2 | ... | ... |
SD3 | SD3 | ||
SD4 | SD4 | ||
SD5 | SD5 | ||
SD6 | SD6 | ||
SD7 | SD7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
SD1 - SD7 | 50 µL | Serial Dilutions (1 to 1000 µM) |
BL | 50 µL | Dilution Buffer |
TS | 50 µL | test sample |
- Prepare D-Lactate standards (SD), 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.
- Add 50 µL of D-Lactate working solution to each well of D-Lactate standard, blank control, and test samples to make the total D-Lactate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of D-Lactate working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, Cutoff = 570 nm).
Images
Citations
Authors: Zhang, Jie and Chen, Zhewen and Lu, Yanwen and Tu, Daoyuan and Zou, Fengqian and Lin, Shouwen and Yu, Weinan and Miao, Mingyong and Shi, Hanping
Journal: OncoTargets and therapy (2021): 1465
Authors: Zhang, Rufeng and Hou, Tingting and Cheng, Heping and Wang, Xianhua
Journal: The FASEB Journal (2019): fj--201901117RR
Authors: Chen, Liping and Mai, Weiqian and Chen, Minfeng and Hu, Jianyang and Zhuo, Zhenjian and Lei, Xueping and Deng, Lijuan and Liu, Junshan and Yao, Nan and Huang, Maohua and others, undefined
Journal: Pharmacological Research (2017)
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Journal: Journal of Medicinal Chemistry (2017)
Authors: Zhang, Xiaofang and Wang, Lei and Zhang, Xiaodong and Ren, Lijun and Shi, Wenjing and Tian, Yijun and Zhu, Jiangbo and Zhang, Tianbao
Journal: Food and Chemical Toxicology (2017)
Authors: Sünwoldt, Juliane and Bosche, Bert and Meisel, Andreas and Mergenthaler, Philipp
Journal: Frontiers in Molecular Neuroscience (2017): 305
Authors: Wang, Dong and Wang, Qingjie and Yan, Gaoliang and Qiao, Yong and Sun, Ling and Zhu, Boqian and Tang, Chengchun and Gu, Yuchun
Journal: Biochemical and biophysical research communications (2015): 607--614
Authors: Nomura, Johji and So, Alex and er , undefined and Tamura, Mizuho and Busso, Nathalie
Journal: The Journal of Immunology (2015): 5718--5724
References
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