Recombinant Cyno/Rhesus Lysyl Oxidase Homolog 2 Protein, CF
Recombinant Cyno/Rhesus Lysyl Oxidase Homolog 2 Protein, CF Summary
Product Specifications
Gln26-Gln774, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
11431-AO
| Formulation | Supplied as a 0.2 μm filtered solution in MES and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM Sodium Borate, 250 mM Urea, 10 mM CaCl2, pH 8.0
- Recombinant Cyno/Rhesus Lysyl Oxidase Homolog 2 (rcynoLOXL2) (Catalog # 11431-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 Fluorescence Read Capability
- Dilute rcynoLOXL2 to 20 µg/mL in Assay Buffer.
- Dilute Benzylamine to 8 mM in Assay Buffer.
- Combine equal volume of 20 µg/mL rcynoLOXL2 and 8 mM Benzylamine. Also create a Substrate Blank by combining equal volumes of Assay Buffer and 8 mM Benzylamine.
- Incubate the reactions for 30 minutes at 37 °C.
- Prepare Substrate Mixture containing 2 units/mL HRP and 40 µM AUR in Assay Buffer.
- Load 50 µL of the incubated reactions into the wells of a black plate and add 50 µL of Substrate Mixture to each well.
- Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively, in endpoint mode. Note: A cut off must be set at a wavelength of 570 nm.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) |
| Incubation time (min) x amount of enzyme (µg) |
- rcynoLOXL2: 0.5 µg
- Benzylamine: 2 mM
- HRP: 1 unit/mL
- Amplex UltraRed: 20 µM
Scientific Data
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2 μg/lane of Recombinant Cynomolgus Monkey/Rhesus Macaque Lysyl Oxidase Homolog 2/LOXL2 His-tag Protein (Catalog # 11431-AO) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 92-102 kDa, under reducing conditions.
Reconstitution Calculator
Background: Lysyl Oxidase Homolog 2/LOXL2
Lysyl Oxidase Homolog 2 (lysyl oxidase-like protein 2, LOXL2) is a member of a five-member lysyl oxidase-like (LOXL) gene family of secreted copper-binding amine oxidases that oxidize primary amine substrates such as collagen and elastin to aldehydes within the extracellular matrix (1, 2). LOXL2 is a secreted protein expressed in many tissues, with elevated levels in reproductive tissues such as placenta, uterus, and prostate (3). The N-terminal region of LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains responsible for protein-protein interactions while the C-terminal region contains the catalytic domain (1, 2). The catalytic domain contains conserved residues required for copper binding, a lysyl tyrosylquinone (LTQ) element required for co-factor formation essential for activity, and a cytokine receptor-like (CRL) domain (2,4). Modulation of the tissue microenvironment implicates a role for LOXL2 in many pathological conditions including fibrosis, atherosclerosis, and tumor development (5). LOXL2 has been shown to promote cell migration and tumor cell invasiveness (6,7) and to enhance tumor progression in various types of cancer including colon, gastric, hepatic, renal cancers (8,9). Dysregulation of LOXL2 has been linked to fibrosis and inflammation (10). Given its role in modulation of the tissue microenvironment involved in pathological conditions, development of inhibitors to LOXL2 is of interest for therapeutic application (2, 9).
- Csiszar, H. (2001) Prog. Nucleic Acid Res. Mol. Biol. 70:1.
- Radic, J. et. al. (2023) Int. J. Mol. Sci. 24:11745.
- Jourdan-Le Saux C. et al. (1999) J. Biol. Chem. 274:12939.
- Maki, J.M. and K.I. Kivirikko (2001) Biochem. J. 355:381.
- Wang, T.H. et. al. (2016) Int. J. Mol. Sci. 18:62.
- Akiri, G. et al. (2003) Cancer Res. 63:1657.
- Hollosi, P. et al. (2009) Int. J. Cancer. 125:318.
- Peinado, H. et al. (2008) Cancer Res. 68:4541.
- Liburkin-Dan, R. et. al. (2022) Int. J. Mol. Sci. 23:6249.
- Poe, A. et. al. (2023) Am. J. Physiol. Cell Physiol. 325:C694.
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