Amplite® Fluorimetric D-Lactate Dehydrogenase (LDH) Assay Kit
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
Fluorescence microplate reader
|Recommended plate||Solid black|
AT A GLANCE
- Prepare D-lactate Dehydrogenase working solution (50 µL)
- Add D-lactate Dehydrogenase standards or test samples (50 µL)
- Incubate at room temperature for 30 minutes - 2 hours
- Monitor fluorescence increase at Ex/Em = 540/590 nm (Cutoff = 570nm)
Thaw one of each kit component at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. NAD stock solution (100X):
Add 100 µL of H2O into the vial of NAD (Component C) to make 100X NAD stock solution.
2. D-LDH standard solution (100 U/mL):
Add 100 µL of H2O or 1x PBS buffer into the vial of D-LDH standard (Component D) to make 100 U/mL D-LDH standard solution.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13808
Add 10 µL of 100 U/mL D-LDH standard solution into 990 µL 1x PBS buffer to generate 1000 mU/mL D-LDH standard solution. Take 1000 mU/mL D-LDH standard solution and perform 1:3 serial dilutions in PBS to get serial dilutions of D-LDH standard (SD7 - SDH1). Note: Diluted D-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 Probe (Component A) to have Enzyme Probe mixture. Note: This Enzyme Probe mixture is enough for two 96-well plate.
2. Add 50 µL of 100X NAD stock solution into 5 mL Enzyme Probe mixture and mix well to make D-LDH working solution. Note: This D-LDH working solution is enough for one 96-well plate. It is not stable - make enough for one experiment and use promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of D-LDH standards and test samples in a solid black 96-well microplate. SD=D-LDH Standards (SD1 - SD7, 0.3 to 300 mU/mL), BL=Blank Control, TS=Test Samples.
Table 2. Reagent composition for each well.
|SD1 - SD7||50 µL||Serial Dilutions (0.3 to 300 mU/mL)|
|BL||50 µL||Dilution Buffer|
|TS||50 µL||test sample|
- Prepare D-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 D-LDH working solution to each well of D-LDH standard, blank control, and test samples to make the total D-LDH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of D-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 fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, Cutoff 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. The absorption detection has lower sensitivity compared to fluorescence reading.
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)
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)
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)
Authors: Sünwoldt, Juliane and Bosche, Bert and Meisel, Andreas and Mergenthaler, Philipp
Journal: Frontiers in Molecular Neuroscience (2017): 305
Authors: Wang, Dong and Wang, Qingjie and Yan, Gaoliang and Qiao, Yong and Sun, Ling and Zhu, Boqian and Tang, Chengchun and Gu, Yuchun
Journal: Biochemical and biophysical research communications (2015): 607--614
Authors: Nomura, Johji and So, Alex and er , undefined and Tamura, Mizuho and Busso, Nathalie
Journal: The Journal of Immunology (2015): 5718--5724
Authors: Prabhakaran R, Kalaivani P, Huang R, Poornima P, Vijaya Padma V, Dallemer F, Natarajan K.
Journal: J Biol Inorg Chem (2013): 233
Authors: Armstrong AJ, George DJ, Halabi S.
Journal: J Clin Oncol (2012): 3402
Authors: Kim JH, Lee J, Sohn HJ, Song HO, Kim JY, Lee WJ, Park H, Shin HJ.
Journal: Parasitol Res (2012): 1645
Authors: Sugai F, Baba K, Toyooka K, Liang WC, Nishino I, Yamadera M, Sumi H, Fujimura H, Nishikawa Y.
Journal: Neuromuscul Disord (2012): 159
Authors: Nieder C, Marienhagen K, Dalhaug A, Norum J.
Journal: ScientificWorldJournal (2012): 609323
Authors: Gao B, Liu FL, Zhao B.
Journal: Eur J Radiol (2012): 2844
Authors: Orjuela-Sanchez P, Duggan E, Nolan J, Frangos JA, Carvalho LJ.
Journal: Malar J (2012): 366
Authors: Singh TD, Barbhuiya MA, Gupta S, Shrivastav BR, Jalaj V, Agarwal N, Tiwari PK.
Journal: Indian J Clin Biochem (2011): 146
Authors: Odet F, Gabel SA, Williams J, London RE, Goldberg E, Eddy EM.
Journal: Biol Reprod (2011): 556
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
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Ameliorative Effect of Novel Vitamin Formula with Herbal Extracts on Scopolamine-Induced Alzheimer's Disease
An Increase in Plasma Homovanillic Acid with Cocoa Extract Consumption Is Associated with the Alleviation of Depressive Symptoms in Overweight or Obese Adults
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity