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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
  1. Prepare lysyl oxidase standards or test samples (50 µL)
  2. Add lysyl oxidase working solution (50 µL)
  3. Incubate at 37°C for 10 - 30 minutes
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm 
Important      Thaw all the kit components at room temperature before starting the experiment.

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.

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

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.
BLBLTSTS
LS1LS1......
LS2LS2......
LS3LS3
LS4LS4
LS5LS5
LS6LS6
LS7LS7
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.
WellVolumeReagent
LS1-LS750 µLSerial Dilution (0.04 to 4 µg/mL)
BL50 µLAssay Buffer (Component B)
TS50 µLTest Sample

Lysyl Oxidase assay in supernatants
  1. 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.
  2. 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.
  3. Incubate the reaction at 37°C for 10 to 30 minutes, protected from light.
  4. 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
  1. 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.
  2. 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.
  3. Incubate the reaction at 37°C for 10 to 30 minutes, protected from light.
  4. 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

Product family

NameExcitation (nm)Emission (nm)
Amplite® Fluorimetric Glucose Oxidase Assay Kit *Red Fluorescence*571584
Amplite® Fluorimetric Glutamate Oxidase Assay Kit *Red Fluorescence*571584
Amplite® Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*571584
Amplite® Fluorimetric Xanthine Oxidase Assay Kit *Red Fluorescence*571584

Citations

View all 55 citations: Citation Explorer
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
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
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
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)

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
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
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
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
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
Page updated on November 23, 2024

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Catalog Number15255
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Request quotation

Spectral properties

Excitation (nm)

571

Emission (nm)

584

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components

Lysyl oxidase dose response was measured with Amplite® Fluorimetric Lysyl Oxidase Assay Kit on a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Lysyl oxidase dose response was measured with Amplite® Fluorimetric Lysyl Oxidase Assay Kit on a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Lysyl oxidase dose response was measured with Amplite® Fluorimetric Lysyl Oxidase Assay Kit on a solid black 96-well plate using a Gemini fluorescence microplate reader (Molecular Devices).
Effect of LOX inhibition on LPS-induced EC inflammatory activation. Human pulmonary EC grown on 2.8(300 &micro;M), and then stimulated with LPS (200 ng/ml) for 48 hrs with or without BAPN. A &ndash; IL-8 production by EC stimulated with or without BAPN was evaluated in conditioned medium by ELISA assay; *P&lt;0.05. B &ndash; Expression of ICAM-1 and VCAM-1 was determined by western blot analysis with specific antibodies. C &ndash; ICAM-1 expression was examined by immunofluorescence staining of stimulated EC using ICAM-1 antibody (green). Counterstaining with DAPI (blue) was used to visualize cell nuclei. D &ndash; HPAEC were transfected with non-specific (nsRNA) or LOX-specific siRNA (si-LOX). ICAM-1 expression was determined by western blot. Equal protein loading was confirmed by membrane re-probing with &beta;-actin antibody. LOX activity and interleukin-8 (IL-8) production in conditioned medium was measured using LOX activity assay (AAT Bioquest, Sunnyvale, CA) and IL-8 ELISA kit (R&amp;D Systems, Minneapolis, MN), respectively, according to the manufacturers' instructions. Source: Graph from <strong>Stiffness-Activated GEF-H1 Expression Exacerbates LPS-Induced Lung Inflammation</strong> by Isa Mambetsariev et al., <em>PLOS</em>, Apr. 2014.
Effects of IL1&beta; and TGF&beta;1 on the expression and activity level of lysyl oxidase in dermal and lung fibroblasts. (A&ndash;B) HDFa and HLFa were treated with IL1&beta;, TGF&beta;1, or a combination of both, for 24 and 48 h. The mRNA levels of LOX were measured with qRT-PCR and expressed as fold change compared to untreated control. (C&ndash;D) Quantification of lysyl oxidase activity as secreted in the culture medium by HDFa and HLFa treated with IL1&beta;, TGF&beta;1, or a combination of both for 24 and 48 h.&nbsp; LOX activity was determined with the Amplite Fluorimetric Lysyl Oxidase Assay Kit (AAT Bioquest Inc, USA) in accordance to the manufacturer's protocol. Source: Graph from <strong>Interleukin-1&beta; Attenuates Myofibroblast Formation and Extracellular Matrix Production in Dermal and Lung Fibroblasts Exposed to Transforming Growth Factor-&beta;1</strong> by Masum M et al., <em>PLOS</em>, Mar. 2014.
Activity of lysyl oxidase (LOX) is increased in whole-lung tissues of chronically hypoxic mice and in culture media from human PASMC exposed to hypoxia or treated with CoCl2. Enzymatic activity of LOX was determined using Fluorimetric Lysyl Oxidase Assay Kit by monitoring LOX-catalyzed H2O2 release from the fluorescent substrate in HRP-coupled reaction. A: LOX activity in lung tissue homogenates from normoxic (Nor, room air for 5 weeks, n = 5) and hypoxic (Hyp, 10% O2 for 5 weeks, n = 5) mice. **P&lt;0.01 vs. Nor. B: LOX activity in culture media collected from human PASMC after 24, 48 and 72 hrs of exposure to normoxia (Nor, 21% O2) or hypoxia (Hyp, 3% O2). *P&lt;0.05, **P&lt;0.01 vs. Nor. C: LOX activity in culture media collected from PASMC treated with vehicle (Cont) or CoCl2 (100 &micro;M for 48 hrs). **P&lt;0.01 vs. Cont. Each bar graph displays the Cu-dependent activity of LOX determined by subtracting values obtained in the presence of BCS (a Cu chelator) from values in the absence of BCS. LOX activity is expressed in nanomoles of H2O2 released from the cells and normalized to the amount of total protein in each sample. Data are shown as mean&plusmn;SE. Activity in the conditioned media and the cell extracts was measured using the Fluorimetric Lysyl Oxidase Assay Kit (AAT Bioquest, Inc., prod. no. 15255). Source: Graph from <strong>Upregulated Copper Transporters in Hypoxia-Induced Pulmonary Hypertension</strong> by Adriana M. Zimnicka et al., <em>PLOS,</em> Mar. 2014.
Atox1 is required for ECM maturation and Cu enzyme LOX activation. (A,B) Masson&rsquo;s Trichrome staining, scale bars&thinsp;=&thinsp;500&thinsp;&mu;m (A) and LOX activity (B) in wound tissues at day 7 after wounding in WT and Atox1<sup>&minus;/&minus;</sup> mice. In (A) boxed regions are shown at higher magnification to the right, scale bars&thinsp;=&thinsp;100&thinsp;&mu;m. Blue color indicates the collagen deposition; light red or pink for keratin, muscle or cytoplasm; and dark brown or black for cell nuclei. W: wound area; WE: wound edge In (B) a graph represents mean&thinsp;&plusmn;&thinsp;SE for LOX activity and a western blot represents Pro-LOX protein expression in wound tissues at days 0 and 7 (n&thinsp;=&thinsp;3. **p&thinsp;&lt;&thinsp;0.01 vs. WT). (C) Schematic diagram showing the essential role of Cu-dependent transcription factor and Cu chaperone function of Atox1 in Cu-dependent wound healing.&nbsp;Source: <strong>Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing <em>in vivo</em></strong> by Das et al., <em>Scientific Reports,</em> Sept. 2016.

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
Amplite® Reagents and Assay Kits
Oxidases
Reactive Oxygen Species (ROS)