Recombinant Human Lysyl Oxidase Homolog 2 Protein, CF

• Assay Buffer: 50 mM Sodium Borate, 1.2 M Urea, 10 mM CaCl₂, pH 8.0 
• Recombinant Human Lysyl Oxidase Homolog 2/LOXL2 (rhLOXL2) (Catalog # 2639-AO)
• Coupling Enzyme: Horseradish Peroxidase (HRP), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
• Substrate Component 1: Benzylamine, 100 mM stock in deionized water
• Substrate Component 2: Amplex Ultra Red (AUR), 10 mM stock in DMSO
• 96-well Black Plate
  
• Plate Reader with Fluorescent Read Capability
  

1. Prepare 50 mM Sodium Borate, 1.2 M Urea, pH 8.0 by dissolving Boric Acid and Urea in deionized water and adjusting the pH with Sodium Hydroxide.  Then, prepare the Assay Buffer by adding 1 M CaCl2 to a final concentration of 10 mM.
2. Dilute rhLOXL2 to 10 ng/µL in Assay Buffer.
3. Dilute Benzylamine to 4 mM in Assay Buffer.
4. Combine equal volumes of 10 ng/µL rhLOXL2 and 4 mM Benzylamine. Also create a Substrate Blank by combining equal volumes of Assay Buffer and 4 mM Benzylamine.
5. Incubate the reactions for 30 minutes at 37 °C.
6. Prepare the Substrate Mixture, 2 units/mL HRP and 40 µM AUR, in Assay Buffer.
7. Load 50 µL of the incubated reactions into the wells of a plate and add 50 µL of Substrate Mixture.
8. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in endpoint mode. Note: A cutoff must be set manually at a wavelength of 570 nm.
9. Calculate specific activity:

     *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 in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

• rhLOXL2: 0.25 µg
• Benzylamine: 1 mM
• HRP: 1 unit/mL
• AUR: 20 µM