Recombinant P. heparinus Heparinase II Protein, CF
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Recombinant P. heparinus Heparinase II Protein, CF Summary
Ala26-Arg772, 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 PBS.|
|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: 100 mM Tris, pH 7.5
- Recombinant P. heparinus Heparinase II (rPhHeparinase II) (Catalog # 6336-GH)
- Substrate: Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
- 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rPhHeparinase II to 20 ng/µL in Assay Buffer.
- Dilute Substrate to 3.0 mg/mL in Assay Buffer.
- Load into a plate 50 µL of the diluted rPhHeparinase II, and start the reaction by adding 50 µL of 3.0 mg/mL Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 3.0 mg/mL Substrate.
- Read in kinetic mode for 5 minutes at an absorbance of 232 nm.
- 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 3800 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rPhHeparinase II: 1.0 µg
- Substrate: 1.5 mg/mL
Heparinase II digestion of Heparin Sulfate (200 μg) is assessed in a 5-minute kinetic assay by monitoring absorbance at 232 nm. R&D SystemsP. heparinusHeparinase I (catalog # 6336-GH) exhibits activity at 2595.6 pm/min/μg.
Background: Heparinase II
Heparan sulfate is a sulfated glycosaminoglycan with the repeating disaccharide units of ‑4HexA1,4GlcNAc beta 1-. It is usually attached to the protein cores of proteoglycans found on cell membrane and extracellular matrix, where it binds to a variety of protein ligands and regulates a wide range of biological activities, including developmental processes, angiogenesis, blood coagulation and tumor metastasis (1, 2). Heparan sulfate has a domain structure containing sulfated regions interspaced with less or non-sulfated regions (3, 4). Heparin shares the backbone structure with heparan sulfate but contains no non-sulfated regions. Heparinases are a family of lyases that release unsaturated oligosaccharides from heparin and heparan sulfate upon digestion (5). Heparinase I recognizes highly sulfated regions and is more specific for heparin. Heparinase II digests both heparin and heparan sulfate. Heparinase III prefers less-sulfated regions and is active only on heparan sulfate (6, 7).
- MacArthur, J. M. et al. (2007) J. Clin. Invest. 117:153.
- Esko, J. D. and Selleck, S. B. (2002) Annu. Rev. Biochem. 71:435.
- Maccarana, M. et al. (1996) J. Biol. Chem. 271:17804.
- Linker, A. and Hovingh, P. (1975) Biochim. Biophys. Acta. 385:324.
- Linker, A. and Hovingh, P. (1965) J. Biol. Chem. 240:3724.
- Su, H. et al. (1996) Appl. Environ. Microbiol. 62:2723.
- Hovingh, P. and Linker, A. (1970) J. Biol. Chem. 245:6170.
Citation for Recombinant P. heparinus Heparinase II Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.
Authors: Wu ZL, Prather B, Ethen CM
Sample Types: Protein
Applications: Enzyme Assay
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