Overview

  • Product name
    Lysyl Oxidase Activity Assay Kit (Fluorometric)
  • Detection method
    Fluorescent
  • Sample type
    Cell culture supernatant, Cell culture extracts, Tissue Extracts
  • Assay type
    Semi-quantitative
  • Sensitivity
    40 ng/well
  • Assay time
    0h 30m
  • Product overview

    Lysyl Oxidase Activity Assay Kit / LOX Activity Assay Kit (Fluorometric) (ab112139) provides a simple method to measure lysyl oxidase (LOX) activity in cell and tissue extracts from mammals and other species, as well as physiological solutions.


    The LOX activity assay protocol uses a proprietary LOX substrate that releases hydrogen peroxide upon transformation by the LOX present in the sample. Hydrogen peroxide is in turn detected using a red fluorescence substrate for HRP-coupled reactions. This leads to increase in fluorescence that can be easily detected at Ex/Em = 540/590 nm in a fluorescence microplate reader.


    This assay is semi-quantitative as it does not contain a LOX standard for calibration. When a known concentration of LOX is used, the assay can detect activity from as low as 40 ng of lysyl oxidase in solution.


    The assay is highly sensitive and its unique detection method eliminates the interference that occurs in certain biological samples.

  • Notes

    Lysyl oxidase (protein-lysine-6-oxidase, LOX, EC 1.4.3.13) is an extracellular copper-dependent enzyme that catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors.

    The activity of Lysyl oxidase in biological samples is traditionally assessed by tritium release end-point assays using radio isotope labeled collagen or elastin substrates.

  • Platform
    Microplate reader

Properties

Associated products

Images

  • Relative LOX activity levels in mouse tissue. Tissue samples were prepared following assay protocol. Protein concentration was determined and samples were diluted 2-30 fold. LOX activity levels were measured after 15 minutes incubation in a fluorometric plate reader at Ex/Em = 535/587 nm. LOX activity levels are relative to background noise (blank control).

  • LOX activity fluorescence (RFU) values vs quantity (µg). Recombinant human LOX2 was serially diluted 3.35-0.04 (1/3) and activity was measured following assay procedure.

  • LOX activity absorbance (OD) values vs quantity (µg). Recombinant human LOX2 was serially diluted 3.35-0.04 (1/3) and activity was measured following assay procedure. Colorimetric detection is approximately 10-times lower than fluorometric.

  • Typical lysyl oxidase (LOX) dose response curve. Known amounts of LOX were added to wells and reaction was run following assay protocol. Fluorescence was measured on a solid black 96-well plate using a Gemini fluiorescence microplate reader (Molecular Devices). Activity from as low as 40 ng/mL of LOX can be detected after 30 minutes incubation.

Protocols

References

This product has been referenced in:
  • Rosell-Garcia T & Rodriguez-Pascual F Enhancement of collagen deposition and cross-linking by coupling lysyl oxidase with bone morphogenetic protein-1 and its application in tissue engineering. Sci Rep 8:10780 (2018). Read more (PubMed: 30018337) »
  • Wang L  et al. Nano-copper-bearing stainless steel promotes fracture healing by accelerating the callus evolution process. Int J Nanomedicine 12:8443-8457 (2017). Read more (PubMed: 29225463) »
See all 11 Publications for this product

Customer reviews and Q&As

1-10 of 39 Abreviews or Q&A

Question
Answer

Theoretically, azide should be compatible with ab112139 Lysyl Oxidase Activity Assay Kit (Fluorometric), though I am sorry we have not directly tested this so cannot provide data to confirm this.

Compounds and buffer components that may interfere with the assay include DTT, glutathione (reduced form: GSH), β-mercaptoethanol, some detergents (such as Brij-35, Tween-20 and NP40), NADH and NADPH.

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Question
Answer

The laborotory uses 500 uM BAPN from TCI, the cat# is A9754, and the cas# is 2079-89-2. In general the laboratory would recommend between 0.1 - 1 mM as most publications use BAPN concentrations in this range. Please see some example references attached.

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Answer

Please note that this kit is not an ELISA kit but an activity assay kit.

With regards to the standard used, when the kit was first developed, the lab used isolated LOX from pig heart, which is not commercial available. The kit is now tested using recombinant human LOXL2 from R&D Systems-

http://www.rndsystems.com/pdf/2639-AO.pdf

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Answer



Frozen samples are suitable for use with this kit.

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Question
Answer

For cell lysates, the cells should be collected and then homogenized in PBS. After spinning down the cells, the supernatant will be used in the assay. 50 uL of supernatant will be added per well of the assay plate, and follow step C.1. in the protocol (page 10).

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Question
Answer

I can confirm that Solid Black micro plate was used in experiment with top-reading instrument.

The clear bottom black and solid black plates can be selected based on experimental set up and instrument.

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Question
Answer


1. Should one use fresh or frozen samples? --Both are OK as long as there is LOX in the sample
2. Are tissue extracts OK?--Yes.

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Answer


1) Regarding the standard serial dilutions, you are correct it should be 0.04 ug to 4 ug. This has been updated in our online protocols, but older copies of the protocol may still incorrectly say 0.04 ng. Please make the standards starting with 0.04 ug.

2) You can use other LOX standard protein, but we suggest using the R&D product as that is what the lab uses. Searching on http://www.biocompare.com may give you other options of LOX standard. When preparing the standards, use PBS with 0.1% BSA or the buffer in your test sample. The assay buffer contains LOX substrate in it, so don't use it for your sample and/or standard dilution.

3) For the microplate, we suggest using Greiner Bio-one cat #655079.

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Answer


I contacted the scientists in the lab about this question, and we have not tested the kit with samples containing protease inhibitors, but theoretically the kit should be compatible with protease inhibitors. It should be fine to add these to your samples prior to use with the kit.

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Answer

Thank you for contacting us.

The LOX activity is expressed as Fluorescent RFU units. Please check the following publication for reference;

http://www.molecular-cancer.com/content/9/1/206

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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1-10 of 39 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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