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

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1010.11e-2100101- Dose-responseData legend Generated with Quest Graph™ L-Lactate Dehydrogenase (mU/mL) Abs 575/Abs 605 Hover mouse to interact
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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200 Tests 13813 $295


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Overview

Ex/Em (nm)575/None
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsAbsorbance microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
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.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

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 min - 2 hours
  4. Monitor absorbance ratio increase at A575nm/A605nm

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

Key parameters
Instrument:Absorbance microplate reader
Absorbance:575/605 nm
Recommended plate:Clear bottom
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. 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.

Preparation of standard solution
L-LDH standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13813

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.

Preparation of working solution

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 one 96-well plate. 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.

Sample experimental protocol

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. 

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 (0.4 to 300 mU/mL)
BL 50 µL Dilution Buffer
TS 50 µL test 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.
Example data analysis and figures

The reading (Abs 575/Abs 605) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate L-Lactate Dehydrogenase samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. 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.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Application Notes
1. AssayWise Letters 2013, Vol 2(1)

Certificate of Analysis