Amplite® Fluorimetric L-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 L-lactate working solution (50 µL)
- Add L-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
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. L-Lactate standard solution (100 mM):
Add 200 µL of H2O or 1x PBS buffer into the vial of L-Lactate Standard (Component D) to make 100 mM L-Lacate standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13814
Add 10 µL of L-Lactate stock solution into 990 µL PBS buffer to generate 1 mM L-Lactate standard solution (Lac7). Take the 1 mM L-Lactate standard solution and perform 1:3 serial dilutions to get serial dilutions of L-Lactate standard (Lac6 - Lac1). Note: Diluted L-Lactate standard solution is unstable and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A). Add 50 µL NAD stock solution (100X) into the bottle of Component A, and mix well. Note: This L-lactate working solution is not stable, and should be used promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of L-Lactate standards and test samples in a solid black 96-well microplate. Lac= L-Lactate Standards (Lac1 - Lac7, 1 µM to 1 mM), BL=Blank Control, TS=Test Samples.
BL | BL | TS | TS |
Lac1 | Lac1 | ... | ... |
Lac2 | Lac2 | ... | ... |
Lac3 | Lac3 | ||
Lac4 | Lac4 | ||
Lac5 | Lac5 | ||
Lac6 | Lac6 | ||
Lac7 | Lac7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
Lac1 - Lac7 | 50 µL | Serial Dilutions (1 µM to 1 mM) |
BL | 50 µL | Dilution Buffer |
TS | 50 µL | Test Sample |
- Prepare L-Lactate standards (Lac), 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 L-Lactate working solution to each well of L-Lactate standard, blank control, and test samples to make the total L-Lactate assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-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, cut off at 570 nm).
Images
Citations
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Authors: Cao, Duo-Yao and Spivia, Weston R and Veiras, Luciana C and Khan, Zakir and Peng, Zhenzi and Jones, Anthony E and Bernstein, Ellen A and Saito, Suguru and Okwan-Duodu, Derick and Parker, Sarah J and others,
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Authors: Rattu, Gurdeep and Khansili, Nishtha and Maurya, Vaibhav Kumar and Krishna, Prayaga M
Journal: Environmental Chemistry Letters (2020): 1--18
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Journal: Human Mutation (2020): 961--972
Authors: Bernardo, Tiffany M
Journal: (2020)
Authors: Wang, Min and Wang, Yuli and Gao, Bingbing and Bian, Yifeng and Liu, Xiaojiang and He, Zhenzhu and Zeng, Yi and Du, Xin and Gu, Zhongze
Journal: ACS applied materials & interfaces (2019)
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