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Amplite® Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit

D-LDH dose response was measured with Amplite® Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.
D-LDH dose response was measured with Amplite® Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.
D-LDH dose response was measured with Amplite® Colorimetric D-Lactate Dehydrogenase (LDH) Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.
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OverviewpdfSDSpdfProtocol


Lactate dehydrogenase (LDH) is an oxidoreductase enzyme that catalyzes the interconversion of pyruvate and lactate. LDH is present in cytosol of a wide variety of organisms, including animals and plants. Cells release LDH into the bloodstream after tissue damage or red blood cell hemolysis. Since LDH is a fairly stable enzyme, it has been widely used to evaluate the presence of damage and toxicity of tissue and cells. Quantification of LDH has a broad range of applications. LDH is also elevated in certain pathological conditions such as cancer. This Amplite® Lactate Dehydrogenase Assay Kit provides an absorption-based method for detecting D-lactate dehydrogenase (D-LDH) in biological samples such as serum, plasma, urine, as well as in cell culture samples. In the enzyme coupled assay, LDH is proportionally related to the concentration of NADH that is specifically monitored by a chromogenic NADH sensor. This assay is specific for D-LDH. The absorption signal can be read by an absorption microplate reader at the absorbance ratio of ~570 nm to ~605 nm (A575 nm/A605 nm). With this colorimetric Amplite® D-lactate Dehydrogenase Assay Kit, we were able to detect as little as 3 mU/mL D-lactate dehydrogenase in a 100 µL reaction volume.

Platform


Absorbance microplate reader

Absorbance575/605 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare D-Lactate Dehydrogenase working solution (50 µL)
  2. Add D-lactate Dehydrogenase standards or test samples (50 µL)
  3. Incubate at room temperature for 30 minutes - 2 hours
  4. 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

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

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

D-LDH standard

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.

BLBLTSTS
SD1SD1......
SD2SD2......
SD3SD3  
SD4SD4  
SD5SD5  
SD6SD6  
SD7SD7  

 

 

Table 2. Reagent composition for each well.

WellVolumeReagent
SD1 - SD750 µLSerial Dilutions (0.3 to 300 mU/mL)
BL50 µLDilution Buffer
TS50 µLtest sample
  1. 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.

  2. 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.

  3. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.

  4. Monitor the absorbance ratio increase with an absorbance plate reader at A575nm/A605nm.

Images


Citations


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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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High glucose and interleukin 1β-induced apoptosis in human umbilical vein endothelial cells involves in down-regulation of monocarboxylate transporter 4
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