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Amplite® Fluorimetric L-Lactate Dehydrogenase (LDH) Assay Kit

L-LDH dose response was measured with Amplite® Fluorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.
L-LDH dose response was measured with Amplite® Fluorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.
L-LDH dose response was measured with Amplite® Fluorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


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 a fluorescence-based method for detecting L-lactate dehydrogenase (L-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 fluorogenic NADH sensor. This assay is specific for L-LDH. The fluorescence signal can be read by a fluorescence microplate reader. With this fluorimetric Amplite® L-lactate Dehydrogenase Assay Kit, we were able to detect as little as 1 mU/mL L-lactate dehydrogenase in a 100 µL reaction volume.


Absorbance microplate reader

Absorbance575/605 nm
Recommended plateSolid white

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black


Example protocol


Protocol summary

  1. Prepare L-lactate dehydrogenase working solution (50 µL)
  2. Add L-lactate dehydrogenase standards or test samples (50 µL)
  3. Incubate at room temperature for 30 minutes - 2 hours
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
Thaw one of each kit component at room temperature before starting the experiment.


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. L-LDH standard solution (100 U/mL):
Add 100 µL of H2O or 1X PBS buffer into the vial of L-LDH standard (Component D) to make 100 U/mL L-LDH standard solution.


L-LDH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13812

Add 10 µL of L-LDH standard solution into 990 µL 1X PBS buffer to generate 1000 mU/mL L-LDH standard solution. Take 1000 mU/mL L-LDH standard solution to perform 1:3 serial dilutions to get serial dilutions of L-LDH standard (L-LDH1 - L-LDH7). Note: Diluted L-LDH standard solution is unstable, and should be used within 4 hours.


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 plates.

2. Add 50 µL NAD stock solution (100X) into 5 mL of enzyme probe mixture, and mix well. Note: This L-LDH working solution is not stable, and should be used promptly.


Table 1. Layout of L-LDH standards and test samples in a solid black 96-well microplate. L-LDH = L-LDH standard (L-LDH1 - L-LDH7, 0.3 to 300 mU/mL); BL = blank control; TS = test sample.


 Table 2. Reagent composition for each well.

L-LDH1 -
50 µLSerial Dilution (0.3 to 300 mU/mL)
BL50 µLDilution Buffer
TS50 µLTest Sample
  1. Prepare L-LDH standards (L-LDH), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of L-LDH working solution to each well of L-LDH standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well isntead, 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 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). Note: The contents of the plate can also be transferred to a white/clear bottom plate and read by absorbance microplate reader at the ratio of A575nm/A605nm. However, the absorption detection will have a lower sensitivity compared to that of the fluorescence reading.



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