Amplite® Colorimetric D-Lactate Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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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 | SDSProtocol |
Platform
Absorbance microplate reader
Absorbance | 575/605 nm |
Recommended plate | Clear bottom |
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 absorbance ratio increase at A575nm/A605nm
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-Lactate standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13811
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. It is unstable and should be used promptly within 2 hours. Avoid exposure to light. Note: Alternatively, one can make a 50X of D-Lactate Enzyme Mix stock solution by adding 100 μL of H2O into the bottle of Enzyme Mix (Component A), and then prepare the D-Lactate working solution by mix the stock solution with Assay Buffer (Component B) and 100X NAD stock solution proportionally.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of D-Lactate standards and test samples in a white clear bottom 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 absorbance ratio increase with an absorbance plate reader at A575nm/A605nm.
Images
Citations
Authors: Yu, Ruyang and Zhou, Qilyu and Liu, Tianlong and Liu, Ping and Li, Hong and Bian, Yifei and Liu, Zhongjie
Journal: Journal of Functional Foods (2023): 105646
Authors: Liu, Wenhan and Zha, Pingping and Guo, Lili and Chen, Yueping and Zhou, Yanmin
Journal: Animal Feed Science and Technology (2023): 115570
Authors: Xie, Ting and Liu, Wenhan and Chen, Yueping and Zhou, Yanmin
Journal: Poultry Science (2022): 102108
Authors: Chen, Yueping and Zha, Pingping and Xu, Hongrui and Zhou, Yanmin
Journal: Poultry Science (2022): 102257
Authors: Zhang, Hao and Chen, Yanan and Chen, Yueping and Li, Yue and Jia, Peilu and Ji, Shuli and Zhou, Yanmin and Wang, Tian
Journal: Journal of animal science (2020): skz373
Authors: Zhang, Hao and Chen, Yanan and Chen, Yueping and Ji, Shuli and Jia, Peilu and Li, Yue and Wang, Tian
Journal: Journal of Animal Science and Biotechnology (2020): 1--16
Authors: Zhang, Hao and Chen, Yanan and Li, Yue and Wang, Tian
Journal: Oxidative medicine and cellular longevity (2020)
Authors: Zhang, Hao and Chen, Yanan and Chen, Yueping and Li, Yue and Jia, Peilu and Ji, Shuli and Zhou, Yanmin and Wang, Tian
Journal: Journal of Animal Science (2019)
Authors: Zou, X and Ji, J and Qu, H and Wang, J and Shu, DM and Wang, Y and Liu, TF and Li, Y and Luo, CL
Journal: Poultry science (2019)
Authors: Di Iorio, Biagio Raffaele and Rocchetti, Maria Teresa and De Angelis, Maria and Cosola, Carmela and Marzocco, Stefania and Di Micco, Lucia and di Bari, Ighli and Accetturo, Matteo and Vacca, Mirco and Gobbetti, Marco and others, undefined
Journal: Journal of clinical medicine (2019): 1424
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FAQ
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