logo
AAT Bioquest

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.
Ordering information
Price
Catalog Number
Unit Size
Quantity
Add to cart
Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

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 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.

Platform


Absorbance microplate reader

Absorbance575/605 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

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.

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

  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.

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.

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

Table 2. Reagent composition for each well.

WellVolumeReagent
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.

Images


Citations


View all 13 citations: Citation Explorer
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
One step open fermentation for lactic acid production from inedible starchy biomass by thermophilic Bacillus coagulans IPE22
Authors: Wang, Yujue and Cao, Weifeng and Luo, Jianquan and Qi, Benkun and Wan, Yinhua
Journal: Bioresource Technology (2018)
Arenobufagin inhibits prostate cancer epithelial-mesenchymal transition and metastasis by down-regulating β-catenin
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, undefined
Journal: Pharmacological Research (2017)
Fibroblast Activation Protein Alpha-Activated Tripeptide Bufadienolide Anti-tumor Prodrug with Reduced Cardiotoxicity
Authors: Deng, Li-Juan and Wang, Long-Hai and Peng, Cheng-Kang and Li, Yi-Bin and Huang, Mao-Hua and Chen, Min-Feng and Lei, Xue-Ping and Qi, Ming and Cen, Yun and Ye, Wen-Cai and others, undefined
Journal: Journal of Medicinal Chemistry (2017)
The use of KnockOut serum replacement (KSR) in three dimentional rat testicular cells co-culture model: An improved male reproductive toxicity testing system
Authors: Zhang, Xiaofang and Wang, Lei and Zhang, Xiaodong and Ren, Lijun and Shi, Wenjing and Tian, Yijun and Zhu, Jiangbo and Zhang, Tianbao
Journal: Food and Chemical Toxicology (2017)
Neuronal Culture Microenvironments Determine Preferences in Bioenergetic Pathway Use
Authors: Sünwoldt, Juliane and Bosche, Bert and Meisel, Andreas and Mergenthaler, Philipp
Journal: Frontiers in Molecular Neuroscience (2017): 305

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
Association of degree and type of edema in posterior reversible encephalopathy syndrome with serum lactate dehydrogenase level: initial experience
Authors: Gao B, Liu FL, Zhao B.
Journal: Eur J Radiol (2012): 2844
A lactate dehydrogenase ELISA-based assay for the in vitro determination of Plasmodium berghei sensitivity to anti-malarial drugs
Authors: Orjuela-Sanchez P, Duggan E, Nolan J, Frangos JA, Carvalho LJ.
Journal: Malar J (2012): 366
Quantitative Assessment of Expression of Lactate Dehydrogenase and its Isoforms 3 and 4 may Serve as Useful Indicators of Progression of Gallbladder Cancer: A Pilot Study
Authors: Singh TD, Barbhuiya MA, Gupta S, Shrivastav BR, Jalaj V, Agarwal N, Tiwari PK.
Journal: Indian J Clin Biochem (2011): 146
Lactate dehydrogenase C and energy metabolism in mouse sperm
Authors: Odet F, Gabel SA, Williams J, London RE, Goldberg E, Eddy EM.
Journal: Biol Reprod (2011): 556
Lactate dehydrogenase, Gleason score and HER-2 overexpression are significant prognostic factors for M1b prostate cancer
Authors: Yamada Y, Nakamura K, Aoki S, Tobiume M, Zennami K, Kato Y, Nishikawa G, Yoshizawa T, Itoh Y, Nakaoka A, Yoshida E, Uchiyama T, Honda N.
Journal: Oncol Rep (2011): 937