Recombinant Human HO-2/HMOX2 Protein, CF Summary
Ser2-Leu291, with a C-terminal 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 Tris and NaCl.|
|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 Tris, pH 7.5
- Recombinant Human HO‑2/HMOX2 (rhHO‑2) (Catalog # 3170-HM)
- Recombinant Human POR/Cytochrome P450 Reductase (rhPOR) (Catalog # 6340-PR)
- Recombinant Human Biliverdin Reductase A/BLVRA (rhBLVRA) (Catalog # 6454-BR)
- Catalase (Sigma, Catalog # C30), 100,000 U/mL stock
- Hemin (Sigma, Catalog # H9039), 10 mM stock in DMSO
- Bovine Serum Albumin (BSA), 100 mg/mL stock in deionized water
- beta -Nicotinamide adenine 2'-phosphate reduced tetrasodium salt hydrate (NADPH) (Sigma, Catalog # N7505), 20 mM stock in deionized water
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute Hemin to 1 mM in Assay Buffer.
- Prepare Reaction Mixture containing 60 µM Hemin, 4 mg/mL BSA, 1000 U/mL catalase, 80 µg/mL rhPOR, and 80 µg/mL rhBLVRA in Assay Buffer.
- Prepare 80 µg/mL rhHO-2 in Assay Buffer.
- Dilute NADPH to 1 mM in Assay Buffer.
- In a clear plate, load 25 µL dilute rhHO-2 and add 25 µL Reaction Mixture. Include an enzyme blank containing 25 µL Assay Buffer and 25 µL Reaction Mixture.
- Start the reaction by adding 50 µL 1 mM NADPH to all wells used.
- Read absorbance at 468 nm (bottom read) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Using the extinction coefficient 43500 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Reaction:
- rhHO-2: 2 µg
- rhPOR: 2 µg
- rhBLVRA: 2 µg
- Catalase: 250 U/mL
- Hemin: 15 µM
- BSA: 1 mg/mL
- NADPH: 0.5 mM
Heme oxygenase (HO) is the rate limiting enzyme in heme catabolism (1). It cleaves heme to biliverdin, carbon monoxide, and iron; biliverdin is subsequently converted by biliverdin reductase to bilirubin. The mechanism of HO is unique in that heme serves as both the substrate of the enzyme and as the prosthetic group for the activation of iron-bound O2. HO activity is highest in spleen where senescent erythrocytes are sequestered and destroyed. Two isoforms, HO-1 and HO-2, are expressed in most tissues. HO-1 is an inducible enzyme in response to heavy metals, oxidative stress, cytokines, and many drugs (2). HO-2 is constitutively expressed in the brain and testes. HO-1 is expressed mainly in spleen, liver, and kidney. In addition to its activity in the heme degradation pathway, HO-2 potentially serves as an oxygen sensor through its heme regulating motifs, which have the core sequence of Cys-Pro (3).
- Nader, G. (2008) Pharmacol. Rev. 60:79.
- Otterbein, L.E. and A.M.K. Choi (2000) Am. J. Physiol. Lung Cell Mol. Physiol. 279:1029.
- Yi. L. et al. (2009) J. Biol. Chem. 284:20556.
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