Amplite® Fluorimetric Lysyl Oxidase Assay Kit *Red Fluorescence*
Lysyl oxidase (LOX) is an extracellular enzyme that catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors. These aldehydes are highly reactive, and undergo spontaneous chemical reactions with other lysyl oxidase-derived aldehyde residues, or with unmodified lysine residues. This results in cross-linking collagen and elastin which is essential for stabilization of collagen fibrils and for the integrity and elasticity of mature elastin. Lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. This kit offers a sensitive fluorescent assay to measure LOX activity using our proprietary LOX substrate that releases hydrogen peroxide upon LOX oxidation. The amount of hydrogen peroxide released by the LOX oxidation is detected using our Amplite® HRP substrate in the HRP-coupled reactions. This method allows the detection of sub ng/mL lysyl oxidase and is much more sensitive than the currently available fluorimetric assay for this enzyme activity. This method eliminates the interference that occurs in some biological samples and can be readily used to detect lysyl oxidase activity in cell culture experiments. Please note that the kit does not include the lysyl oxidase enzyme.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare lysyl oxidase standards or test samples (50 µL)
- Add lysyl oxidase working solution (50 µL)
- Incubate at 37°C for 10 - 30 minutes
- Monitor fluorescence intensity at Ex/Em = 540/590 nm
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
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.
Note Amplite™ HRP Substrate is unstable in the presence of thiols such as DTT, glutathione (reduced form: GSH) and β-mercaptoethanol. The presence of thiols at concentration higher than 10 µM will significantly decrease the assay dynamic range. Some detergents (such as Brij-35, Tween-20 and NP40), NADH, and NADPH can also interfere with this assay.
1. Amplite™ HRP Substrate stock solution (250X)
Add 100 µL of DMSO (Component D) into the vial of Amplite™ HRP Substrate (Component A).Note Amplite™ HRP Substrate is unstable in the presence of thiols such as DTT, glutathione (reduced form: GSH) and β-mercaptoethanol. The presence of thiols at concentration higher than 10 µM will significantly decrease the assay dynamic range. Some detergents (such as Brij-35, Tween-20 and NP40), NADH, and NADPH can also interfere with this assay.
2. Horseradish Peroxidase stock solution (50 U/mL)
Add 1 mL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/15255
https://www.aatbio.com/tools/serial-dilution/15255
Lysyl Oxidase standard
Prepare lysyl oxidase standards by serial dilution to obtain standards from 0.04 to 4 µg/mL (LS1 - LS7).Note: Lysyl oxidase standard is not provided in this kit. It can be purchased from R&D Sytems (2639-AO-010 or 6069-AO-010).PREPARATION OF WORKING SOLUTION
Amplite™ HRP Substrate working solution
Add 20 μL of Amplite™ HRP Substrate stock solution (250X) and 20 μL of Horseradish Peroxidase (50 U/mL) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.04 mL.Note The working solution is not stable, use it promptly and avoid direct exposure to light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of lysyl oxidase standards and test samples in a solid black 96-well microplate. LS = lysyl oxidase standard (LS1-LS7, 0.04 to 4 µg/mL); BL = blank control; TS = test sample.
Table 2. Reagent composition for each well. Note that high concentration of Lysyl Oxidase may cause reduced fluorescence signal due to the over oxidation of Amplite™ HRP Substrate (to a non-fluorescent product). Lysyl Oxidase standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.
BL | BL | TS | TS |
LS1 | LS1 | ... | ... |
LS2 | LS2 | ... | ... |
LS3 | LS3 | ||
LS4 | LS4 | ||
LS5 | LS5 | ||
LS6 | LS6 | ||
LS7 | LS7 |
Well | Volume | Reagent |
LS1-LS7 | 50 µL | Serial Dilution (0.04 to 4 µg/mL) |
BL | 50 µL | Assay Buffer (Component B) |
TS | 50 µL | Test Sample |
Lysyl Oxidase assay in supernatants
- Prepare lysyl oxidase standards (LS), blank controls (BL) and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of each corresponding reagent instead of 50 µL.
- Add 50 µL of lysyl oxidase working solution into each well of lysyl oxidase standard, blank control, and test samples to make the total lysyl oxidase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well for a total volume of 50 µL.
- Incubate the reaction at 37°C for 10 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm.
Note The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. Note though that the absorption detection has lower sensitivity compared to fluorescence reading.
Lysyl Oxidase assay for cells
- Prepare cells in a 96-well plate (50 - 100 µL/well), and activate the cells as desired. For a 384-well plate, use 25 µL/well instead. Harvest the cell media.
Note The negative controls (media alone and non-activated cells) are included for measuring background fluorescence. - Add 50 µL of lysyl oxidase working solution into each well of the cell media (from previous step) and well of lysyl oxidase standards (see Table 1). For a 384-well plate, add 25 µL of working solution into each well instead.
- Incubate the reaction at 37°C for 10 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 530 to 570/590 to 600 nm (maximum Ex/Em = 540/590 nm, cut off 570 nm).
Spectrum
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Citations
View all 55 citations: Citation Explorer
Effects of estrogen, progesterone, and relaxin on gene expression and collagen remodeling enzyme activities in primary human tendon cells
Authors: Tuin, Ho Shun
Journal: (2024)
Authors: Tuin, Ho Shun
Journal: (2024)
HCG supplement did not accelerate tunica albuginea remodeling to facilitate penile growth
Authors: Li, Tao and Tian, Yuan and Zhong, Quliang and Chen, Peng and Zhang, Junhao and Du, Guangshi and Li, Lei and Jiang, Yiting and Jiang, Kehua
Journal: Scientific Reports (2023): 16519
Authors: Li, Tao and Tian, Yuan and Zhong, Quliang and Chen, Peng and Zhang, Junhao and Du, Guangshi and Li, Lei and Jiang, Yiting and Jiang, Kehua
Journal: Scientific Reports (2023): 16519
Triptolide attenuates pulmonary fibrosis by inhibiting fibrotic extracellular matrix remodeling mediated by MMPs/LOX/integrin
Authors: Lin, Weiji and Song, Yaqin and Li, Tingting and Yan, Jiahui and Zhang, Ruiyuan and Han, Liang and Ba, Xin and Huang, Yao and Qin, Kai and Chen, Zhe and others,
Journal: Biomedicine \& Pharmacotherapy (2023): 115394
Authors: Lin, Weiji and Song, Yaqin and Li, Tingting and Yan, Jiahui and Zhang, Ruiyuan and Han, Liang and Ba, Xin and Huang, Yao and Qin, Kai and Chen, Zhe and others,
Journal: Biomedicine \& Pharmacotherapy (2023): 115394
Lysyl oxidase-like 2 processing by factor Xa modulates its activity and substrate preference
Authors: Wang, Huilei and Poe, Alan and Martinez Yus, Marta and Pak, Lydia and Nandakumar, Kavitha and Santhanam, Lakshmi
Journal: Communications Biology (2023): 375
Authors: Wang, Huilei and Poe, Alan and Martinez Yus, Marta and Pak, Lydia and Nandakumar, Kavitha and Santhanam, Lakshmi
Journal: Communications Biology (2023): 375
Androgen supplement did not accelerate tunica albuginea remodeling to facilitate penile growth
Authors: Sun, Fa and Li, Tao and Jiang, Yiting and Jiang, Kehua and Tian, Ye and Wang, Zhen and Ban, Yong and Gu, Jiang
Journal: (2022)
Authors: Sun, Fa and Li, Tao and Jiang, Yiting and Jiang, Kehua and Tian, Ye and Wang, Zhen and Ban, Yong and Gu, Jiang
Journal: (2022)
References
View all 21 references: Citation Explorer
Overexpression of lysyl oxidase to increase matrix crosslinking and improve tissue strength in dermal wound healing
Authors: Lau YK, Gobin AM, West JL.
Journal: Ann Biomed Eng (2006): 1239
Authors: Lau YK, Gobin AM, West JL.
Journal: Ann Biomed Eng (2006): 1239
Cellular fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic activation
Authors: Fogelgren B, Polgar N, Szauter KM, Ujfaludi Z, Laczko R, Fong KS, Csiszar K.
Journal: J Biol Chem (2005): 24690
Authors: Fogelgren B, Polgar N, Szauter KM, Ujfaludi Z, Laczko R, Fong KS, Csiszar K.
Journal: J Biol Chem (2005): 24690
The Pro-regions of lysyl oxidase and lysyl oxidase-like 1 are required for deposition onto elastic fibers
Authors: Thomassin L, Werneck CC, Broekelmann TJ, Gleyzal C, Hornstra IK, Mecham RP, Sommer P.
Journal: J Biol Chem (2005): 42848
Authors: Thomassin L, Werneck CC, Broekelmann TJ, Gleyzal C, Hornstra IK, Mecham RP, Sommer P.
Journal: J Biol Chem (2005): 42848
A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progression
Authors: Peinado H, Del Carmen Iglesias-de la Cruz M, Olmeda D, Csiszar K, Fong KS, Vega S, Nieto MA, Cano A, Portillo F.
Journal: Embo J (2005): 3446
Authors: Peinado H, Del Carmen Iglesias-de la Cruz M, Olmeda D, Csiszar K, Fong KS, Vega S, Nieto MA, Cano A, Portillo F.
Journal: Embo J (2005): 3446
Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues
Authors: Maki JM, Sormunen R, Lippo S, Kaarteenaho-Wiik R, Soininen R, Myllyharju J.
Journal: Am J Pathol (2005): 927
Authors: Maki JM, Sormunen R, Lippo S, Kaarteenaho-Wiik R, Soininen R, Myllyharju J.
Journal: Am J Pathol (2005): 927
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