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

Name: Amplite® Colorimetric L-Lactate Dehydrogenase (LDH) Assay Kit
Brand: AAT Bioquest
SKU: 13813
Price: 341 USD
Availability: InStock

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.

Example protocol

AT A GLANCE

Protocol Summary

Prepare L-Lactate Dehydrogenase standards or test samples (50 µL)
Add L-Lactate Dehydrogenase working solution (50 µL)
Incubate at room temperature for 30 min - 2 hours
Monitor absorbance ratio increase at A_575nm/A_605nm

Important Note

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

PREPARATION OF STOCK SOLUTIONS

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.

PREPARATION OF STANDARD SOLUTIONS

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

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.

PREPARATION OF WORKING SOLUTION

Add 10 mL of Assay Buffer (Component B) into the bottle of Enzyme Mix (Component A), and mix well.
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.

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

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.
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.
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 A_575nm/A_605nm.

Citations

View all 13 citations: Citation Explorer

The Role of the ArcA Metabolic Regulator in Gram-Negative Bacteremia
Authors: Brown, Aric
Journal: (2023)

METTL5 stabilizes c-Myc by facilitating USP5 translation to reprogram glucose metabolism and promote hepatocellular carcinoma progression
Authors: Xia, Peng and Zhang, Hao and Lu, Haofeng and Xu, Kequan and Jiang, Xiang and Jiang, Yuke and Gongye, Xiangdong and Chen, Zhang and Liu, Jie and Chen, Xi and others,
Journal: Cancer Communications (2023)

Biophysical and Biochemical Characterization of TP0037, a d-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum
Authors: Deka, Ranjit K and Liu, Wei Z and Norgard, Michael V and Brautigam, Chad A
Journal: Mbio (2020): e02249--20

Activation of Melatonin Receptor 2 But Not Melatonin Receptor 1 Mediates Melatonin-conferred Cardio-protection Against Myocardial Ischemia/Reperfusion Injury
Authors: Han, Dong and Wang, Yong-Jun and Chen, Jiangwei and Zhang, Jibin and Yu, Peng and Zhang, Ran and Li, Shuang and Tao, Bo and Wang, Yabin and Qiu, Ya and others, undefined
Journal: Journal of pineal research (2019): e12571

Mechanisms behind resistance to PI3K inhibitor treatment induced by the PIM kinase
Authors: Song, Jin H and Singh, Neha and Luevano, Libia A and Padi, Sathish KR and Okumura, Koichi and Olive, Virginie and Black, Stephen M and Warfel, Noel A and Goodrich, David W and Kraft, Andrew S
Journal: Molecular cancer therapeutics (2018): 2710--2721

References

View all 72 references: Citation Explorer

DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography
Authors: Prabhakaran R, Kalaivani P, Huang R, Poornima P, Vijaya Padma V, Dallemer F, Natarajan K.
Journal: J Biol Inorg Chem (2013): 233

Serum lactate dehydrogenase predicts for overall survival benefit in patients with metastatic renal cell carcinoma treated with inhibition of mammalian target of rapamycin
Authors: Armstrong AJ, George DJ, Halabi S.
Journal: J Clin Oncol (2012): 3402

Production of monoclonal antibodies for Plasmodium vivax lactate dehydrogenase and patient sera screening using sandwich ELISA
Authors: Kim JH, Lee J, Sohn HJ, Song HO, Kim JY, Lee WJ, Park H, Shin HJ.
Journal: Parasitol Res (2012): 1645

Adult-onset multiple acyl CoA dehydrogenation deficiency associated with an abnormal isoenzyme pattern of serum lactate dehydrogenase
Authors: Sugai F, Baba K, Toyooka K, Liang WC, Nishino I, Yamadera M, Sumi H, Fujimura H, Nishikawa Y.
Journal: Neuromuscul Disord (2012): 159

Towards improved prognostic scores predicting survival in patients with brain metastases: a pilot study of serum lactate dehydrogenase levels
Authors: Nieder C, Marienhagen K, Dalhaug A, Norum J.
Journal: ScientificWorldJournal (2012): 609323

Page updated on November 21, 2024

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