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Lysyl Oxidase Activity Assay Kit (Fluorometric) (ab112139) provides a simple method to measure lysyl oxidase (LOX) activity in cell and tissue extracts, as well as physiological solutions. The assay 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 our proprietary 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.
Please be aware that 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 much more sensitive than the other assays currently available in the market and its unique detection method eliminates the interference that occurs in certain biological samples.
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Lysyl oxidase (protein-lysine-6-oxidase, LOX, EC 18.104.22.168) is an extracellular copper-dependent 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. The chemical reactions result 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 inhibition can cause osteolathyrism, while its upregulation by tumor cells may promote metastasis of an existing tumor, which makes LOX and important oncological target.
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.
|Assay Buffer||1 x 50ml|
|DMSO||1 x 200µl|
|Horseradish Peroxidase||1 vial|
|HRP Substrate||1 vial|
Our Abpromise guarantee covers the use of ab112139 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent concentration.|
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.
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