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

L-LDH dose response was measured with Amplite® Colorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.
L-LDH dose response was measured with Amplite® Colorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.
L-LDH dose response was measured with Amplite® Colorimetric L-Lactate Dehydrogenase Assay Kit in a 96-well white wall/clear bottom plate using a SpectraMax Plus (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 absoption-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 chromogenic NADH sensor. This assays is specific for L-LDH. The absorption signal can be read by an absorption microplate reader an absorbance ratio of A575nm/A605nm. With this colorimetric Amplite® L-lactate Dehydrogenase Assay Kit, we were able to detect as little as 3 mU/mL L-lactate dehydrogenase in a 100 µL reaction volume.


Absorbance microplate reader

Absorbance575/605 nm
Recommended plateClear bottom


Example protocol


Protocol Summary
  1. Prepare L-Lactate Dehydrogenase standards or test samples (50 µL)
  2. Add L-Lactate Dehydrogenase working solution (50 µL)
  3. Incubate at room temperature for 30 min - 2 hours
  4. Monitor absorbance ratio increase at A575nm/A605nm
Important Note

Thaw one vial 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

NAD stock solution (100X)

Add 100 µL of H2O into the vial of NAD (Component C) to make 100X NAD stock solution.

L-Lactate Dehydrogenase (L-LDH) standard solution (100 U/mL)

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


For convenience, use the Serial Dilution Planner:

L-LDH standard
Add 10 µL of 100 U/mL 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 and perform 1:3 serial dilutions in 1x PBS buffer to get serially diluted L-LDH standards (SD7 - SD1). 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 Mix (Component A), and mix well.
  2. Add 100 µL of 100X NAD stock solution into the bottle of Component A+B and mix well to make L-LDH working solution.

    Note        This L-LDH working solution is enough for two 96-well plates. It is unstable and should be used promptly within 2 hours. Avoid exposure to light. 

    Note        Alternatively, one can make a 50X of L-LDH Enzyme Mix stock solution by adding 200 μL of H2O into the bottle of Enzyme Mix (Component A), and then prepare the L-LDH working solution by mix the stock solution with Assay Buffer (Component B) and 100X NAD stock solution proportionally.


Table 1. Layout of L-LDH standards and test samples in a white clear bottom 96-well microplate. SD= L-LDH Standards (SD1 - SD7, 0.4 to 300 mU/mL), BL=Blank Control, TS=Test Samples.


Table 2. Reagent composition for each well.

SD1 - SD750 µLSerial Dilutions (0.4 to 300 mU/mL)
BL50 µLDilution Buffer
TS50 µLtest sample
  1. Prepare L-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 L-LDH working solution to each well of L-LDH standard, blank control, and test samples to make the total L-LDH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of L-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.



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