Amplite® Colorimetric D-Lactate Dehydrogenase (LDH) 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 | ![]() ![]() |
Platform
Absorbance microplate reader
Absorbance | 575/605 nm |
Recommended plate | Clear bottom |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare D-Lactate Dehydrogenase working solution (50 µL)
- Add D-lactate Dehydrogenase standards or test samples (50 µL)
- Incubate at room temperature for 30 minutes - 2 hours
- Monitor absorbance ratio increase at A575nm/A605nm
Important notes
Thaw one 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-LDH standard solution (100 U/mL):
Add 100 µL of H2O or 1x PBS buffer into the vial of D-LDH standard (Component D) to make 100 U/mL D-LDH standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13809
Add 10 µL of 100X D-LDH standard solution into 990 µL 1x PBS buffer to generate 1000 mU/mL D-LDH standard solution. Take 1000 mU/mL D-LDH standard solution and perform 1:3 serial dilutions in PBS to get serial dilutions of D-LDH standard (SD7 - SD1). Note: Diluted D-LDH standard solution is unstable, and should be used within 4 hours.
PREPARATION OF WORKING SOLUTION
1. Add 10 mL of Assay Buffer (Component B) into the bottle of Enzyme Probe (Component A) to have Enzyme Probe mixture. Note: This Enzyme Probe mixture is enough for two 96-well plate. It is unstable at room temperature and should be used promptly within 2 hours and avoid exposure to light. Alternatively, one can make a 50X of D-LDH Enzyme Mixture stock solution by adding 200 µL of H2O into the bottle of Component A, and then prepare the D-LDH working solution by mix the stock solution with assay buffer (Component B) and 100x NAD solution proportionally.
2. Add 50 µL of 100X NAD stock solution into 5 mL enzyme probe mixture and mix well to make D-LDH working solution. Note: This D-LDH working solution is enough for one 96-well plate. It is not stable - make enough for one experiment and use promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of D-LDH standards and test samples in a white/clear bottom 96-well microplate. SD = D-LDH Standards (SD1 - SDH7, 0.3 to 300 mU/mL), 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 (0.3 to 300 mU/mL) |
BL | 50 µL | Dilution Buffer |
TS | 50 µL | test sample |
- Prepare D-LDH 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-LDH working solution to each well of D-LDH standard, blank control, and test samples to make the total D-LDH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of D-LDH 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: AlMalki, Waleed Hassan and Shahid, Imran
Journal: Journal of Pharmacy \& Pharmacognosy Research (2020): 316--326
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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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,
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Authors: Kadir, Lavin Dlshad
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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: Nguyen, Thu Huong Thi
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Authors: Nomura, Johji and So, Alex and er , undefined and Tamura, Mizuho and Busso, Nathalie
Journal: The Journal of Immunology (2015): 5718--5724
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