Recombinant Human Hydroxyacid Oxidase-1/HAO-1 Protein, CF
Recombinant Human Hydroxyacid Oxidase-1/HAO-1 Protein, CF Summary
Leu2-Ile370, with an N-terminal Met and 6-His tag
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM HEPES, pH 8.0
- Recombinant Human Hydroxyacid Oxidase‑1/HAO‑1 (rhHAO-1) (Catalog # 6197-GO)
- 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: Sodium Glyoxylate (Sigma, Catalog # G4502), 400 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
- Dilute rhHAO-1 to 0.5 ng/µL in Assay Buffer.
- Prepare the Substrate Mixture, 4 mM Sodium Glyoxylate, 2 units/mL HRP and 100 µM AUR, in Assay Buffer.
- In a plate, load 50 µL of 0.5 ng/µL rhHAO-1, and start the reaction by adding 50 µL of the Substrate Mixture (step 2). 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 5 minutes. 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 50 µM AUR, 1 unit/mL HRP, 2 mM Sodium Glyoxylate, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.
- rhHAO-1: 0.025 µg
- Sodium Glyoxylate: 2 mM
- HRP: 1 unit/mL
- AUR: 50 µM
Background: Hydroxyacid Oxidase-1/HAO-1
Glycolate oxidase is a member of the superfamily of the alpha ‑hydroxy acid oxidases (HAO), enzymes that are present in both plants and animals (1). It catalyzes the FMN-mediated oxidation of glycolate to glyoxylate and glyoxylate to oxalate with reduction of oxygen to hydrogen peroxide (2, 3). The co‑factor, FMN, is tightly bound but not covalently linked to the protein. In humans and other vertebrates, HAOs are found primarily in the peroxisomes of liver, kidney, and pancreas. Three HAOs have been identified in humans (4). HAO-1 is most highly expressed in liver and pancreas and is most active on two-carbon substrates such as glycolate. HAO-2 is expressed in liver and kidney and has greater activity against long-chain alpha ‑hydroxy acid substrates such as 2‑hydroxypalmitate. HAO-3 is expressed primarily in the pancreas. Recently, HAO has been identified as a major contributor to hyperoxaluria, a disorder in which large deposits of calcium oxalate form kidney stones (5).
- Caroline, V. et al. (2007) Arch. Biochem. Biophys. 465:410.
- Murray, M.S. et al. (2008) Biochemistry, 47:2439.
- Pennati, A. and G. Gadda. (2009) J. Biol. Chem. 284:31214.
- Jones, J.M. et al. (2000) J. Biol. Chem. 275:12590.
- Monico, G.C. et al. (2002) Kidney Int. 62:392.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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