>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
<1.0 EU per 1 μg of the protein by the LAL method.
82 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
90-100 kDa, reducing conditions
Read Publication using 6069-AO in the following applications:
Substrate Component 1: Benzylamine (Sigma, Catalog # B5136), 100 mM stock in deionized water
Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Preheat plate reader to 37 °C.
Dilute rhLOXL3 to 20 ng/µL in Assay Buffer.
Prepare the Substrate Mixture, 2 mM Benzylamine, 2 units/mL HRP and 40 µM AUR, in Assay Buffer.
In a plate, load 50 µL of 20 ng/µL rhLOXL3, and start the reaction by adding 50 µL of the Substrate Mixture (step 3). Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of the Substrate Mixture.
Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode for 8 minutes at 37 °C. Incorporate a 3 minute lag time for data analysis. Note: A cutoff must be set manually at a wavelength of 570 nm.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)
*Adjusted for Substrate Blank **Derived using a fluorescent standard prepared by incubating 20 µM AUR, 1 unit/mL HRP, 1 mM Benzylamine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.
rhLOXL3: 1.0 µg
Benzylamine: 1 mM
HRP: 1 unit/mL
AUR: 20 µM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Lysyl Oxidase Homolog 3 Protein, CF
Lysyl Oxidase Homolog 3
lysyl oxidase-like 3
Lysyl oxidase-like protein 3
Lysyl oxidase-like protein 3 (LOXL3) is a secreted copper amine oxidase. The N-terminal region of LOXL3 contains four scavenger receptor cysteine-rich (SRCR) domains. The C-terminal region consists of a catalytic domain similar to other lysyl oxidases and a cytokine receptor-like domain (1). The catalytic domain contains conserved residues required for copper binding and formation of a lysyl tyrosylquinone cofactor (2). LOXL3 is expressed primarily in epidermis, kidney, aorta, and chondrocytes (3). The enzyme is expressed at low levels in most other tissues. Like lysyl oxidase, LOXL3 oxidizes the lysine epsilon -amino groups of a number of collagen substrates, and is also active against other primary amines (4). However, LOXL3 does not require proteolytic processing to be activated.
Csiszar, K. (2001) Prog. Nucl. Acid Res. Mol. Biol. 70:1.
Maki, J.M. and K.I. Kivirikko (2001) Biochem J. 355:381.
Molnar, J. et al. (2003) Biochim. Biophys. Acta 1647:220.
Lee, J. and Y. Kim (2006) J. Biol. Chem. 281:37282.
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