Recombinant Human Lysyl Oxidase Homolog 3 Protein, CF

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Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human Lysyl Oxidase Homolog 3 Protein, CF Summary

Details of Functionality
Measured by its ability to produce hydrogen peroxide during the oxidation of benzylamine. The specific activity is >1.5 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Lysyl Oxidase Homolog 3/LOXL3 protein
Met1-Ile753, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Ser26
Structure / Form
Monomer
Protein/Peptide Type
Recombinant Enzymes
Gene
LOXL3
Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
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.
SDS-PAGE
90-100 kDa, reducing conditions
Publications
Read Publication using
6069-AO in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in MES and NaCl.
Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
  • Assay Buffer: 50 mM Sodium Borate, 1.2 M Urea, pH 8.0 
  • Recombinant Human Lysyl Oxidase Homolog 3 (rhLOXL3) (Catalog # 6069-AO)
  • Coupling Enzyme: Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  • 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
  1. Preheat plate reader to 37 °C.
  2. Dilute rhLOXL3 to 20 ng/µL in Assay Buffer.
  3. Prepare the Substrate Mixture, 2 mM Benzylamine, 2 units/mL HRP and 40 µM AUR, in Assay Buffer.
  4. 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.
  5. 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.
  6. 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.

Per Well:
  • rhLOXL3: 1.0 µg
  • Benzylamine: 1 mM
  • HRP: 1 unit/mL
  • AUR: 20 µM

Notes

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

  • EC 1.4.3
  • EC 1.4.3.-
  • EC 1.4.3.13
  • LOL3
  • LOXL
  • LOXL3
  • Lysyl Oxidase Homolog 3
  • lysyl oxidase-like 3
  • Lysyl oxidase-like protein 3

Background

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.
  1. Csiszar, K. (2001) Prog. Nucl. Acid Res. Mol. Biol. 70:1.
  2. Maki, J.M. and K.I. Kivirikko (2001) Biochem J. 355:381.
  3. Molnar, J. et al. (2003) Biochim. Biophys. Acta 1647:220.
  4. Lee, J. and Y. Kim (2006) J. Biol. Chem. 281:37282.

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Publications for Lysyl Oxidase Homolog 3/LOXL3 (6069-AO)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: EnzAct.


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Bioinformatics

Gene Symbol LOXL3
Entrez
Uniprot