Recombinant Human Active Heparanase/HPSE Protein, CF
Recombinant Human Active Heparanase/HPSE Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Gln36-Ile543 with N-terminal 6-His tag
The linker peptide from Ser110-Gln157 is removed by protease treatment
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.
7570-GH
Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and E64. |
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.
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Assay Procedure
- Assay Buffer: 50 mM Sodium Acetate, pH 5.0
- Recombinant Human HPSE (rhHPSE) (7570-GH)
- HPSE Substrate (Catalog # ES020)
- Human Syndecan-4 DuoSet Kit (Catalog # DY2918)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
And the following materials that are routinely used in ELISA:
- Coating Buffer (Catalog # DY006)
- Wash Buffer (25X) (Catalog # WA126)
- Reagent Diluent (10X) (Catalog # DY995)
- Substrate Reagent Pack (Catalog # DY999)
- Stop Solution (Catalog # DY994)
- Prepare an ELISA plate by following the DuoSet kit protocol.
- Dilution factor determination:
a. Dilute the HPSE Substrate stock 100-fold in Reagent Diluent (this will be the first dilution point).
b. Further prepare a 2-fold serial dilution series of the above diluted HPSE Substrate with Reagent Diluent for 6 points.
c. Load 100 µL of each point onto the prepared ELISA plate in duplicate. Load 100 µL of Reagent Diluent to 2 separate wells for blank control.
d. Cover the plate and incubate at room temperature for 2 hours.
e. Follow the DuoSet Assay Procedure from step 4 to step 9 to complete the assay.
f. Determine the dilution factor (n) that achieves an OD between 1.8-3.0. - rhHPSE Activity Detection:
a. Dilute the HPSE Substrate stock by n/10-fold in Assay Buffer, diute rhHPSE to 2 μg/mL in assay buffer.
b. Combine 10 µL of rhHPSE with 10 µL of the diluted HPSE Substrate in a vial. For negative control, combine 10 µL of Assay Buffer and 10 µL of the diluted HPSE Substrate in a vial.
c. Incubate reactions and negative control at 37 °C for 2 hours.
d. After incubation, heat all reactions and negative control at 95 °C for 2 minutes to inactivate rhHPSE.
e. Add 220 µL of Reagent Diluent to each reaction and negative control. Mix well.
f. Load 100 µL of each sample onto the prepared ELISA plate in duplicate.
g. Cover the plate and incubate for 2 hours at room temperature.
h. Follow the DuoSet Assay Procedure form step 4 to step 9 to complete the assay. - Calculate % OD reduction compared with the negative control:
[1-(OD of rhHPSE sample/OD of negative control)] x 100 = % OD reduction
Per Reaction:rhHPSE: 20 ng
Scientific Data
Recombinant Human Active Heparanse/HPSE Protein, CF (Catalog # 7570-GH) selectively cleaves the linkage between a GlcA and an N-sulfo GlcNAc (NS) carrying either a 3-O-sulfo or a 6-O-sulfo group. Can also cleave the linkage between a GlcA and an N-sulfo GlcNAc carrying a 2-O-sulfo group (2S), but not linkages between a GlcA unit and a 2-O-sulfated iduronic acid moiety as depicted in the diagram.
2 μg/lane of Recombinant Human Active Heparanase/HPSE Protein (Catalog # 7570-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 50-60 kDa & 7-9 kDa under reducing conditions.
Reconstitution Calculator
Background: Heparanase/HPSE
Heparanase (HPSE) selectively cleaves heparan sulfate at specific sites on heparan sulfate proteoglycans (HSPGs) (1, 2, 3, 4). The enzyme is synthesized as an inactive 65 kDa proenzyme that is secreted via the Golgi apparatus and associates with the cell membrane through interaction with HSPGs (5). It is then endocytosed and transferred to lysosomes (6) where cathepsin L activates it by removing an internal inhibitory peptide, forming a heterodimer composed of an 8 kDa and a 50 kDa subunit (7, 8). Under certain stimuli, the active enzyme is transferred back to the cell surface, where it participates in extracellular matrix degradation and remodeling (9). HPSE facilitates cell migration associated with metastasis, wound healing and inflammation (10). An increase in its activity is associated with an increase in VEGF activity, which further enhances angiogenesis (11). HPSE also enhances shedding of syndecans and increases endothelial invasion and angiogenesis in myelomas (12). It acts as a procoagulant by increasing the generation of activation factor X in the presence of tissue factor and activation factor VII (13). In addition, it increases cell adhesion to the extracellular matrix (ECM), independent of its enzymatic activity (14). HPSE is highly expressed in placenta and spleen and weakly expressed in lymph node, thymus, peripheral blood leukocytes, bone marrow, endothelial cells, fetal liver and tumor tissues (15). The enzyme activity of recombinant human HPSE was assayed in an ELISA format using non-reducing end biotinylated heparan sulfate on recombinant syndecan 4 as a substrate (16).
- Vlodavsky, I. et al. (1999) Nat. Med. 5:793.
- Hulett, M.D. et al. (1999) Nat. Med. 5:803.
- Gong, F. et al. (2003) J. Biol. Chem. 278:35152.
- Peterson, S.B. and Liu, J. (2010) J. Biol. Chem. 285:14504.
- Nadav L. et al. (2002) J. Cell Sci. 115:2179.
- Gingis-Velitski, S. et al. (2004) J. Biol. Chem. 279:44084.
- Abboud-Jarrous, G. et al. (2008) J. Biol. Chem. 283:18167.
- Zetser, A. et al. (2004) J. Cell Sci. 117:2249.
- Zcharia E. et al. (2001) J. Mammary Gland Biol. Neoplasia 6:311.
- Fux, L. et al. (2009) Trends Biochem. Sci. 34:511.
- Cohen-Kaplan, V. et al. (2008) Int. J. Cancer 123:2566.
- Purushothaman, A. et al. (2010) Blood 115:2449.
- Nadir, Y. et al. (2010) Haematologica 95:1927.
- Goldshmidt, O. et al. (2003) FASEB J. 17:1015.
- Kussie, P.H. et al. (1999) Biochem. Biophys. Res. Commun. 261:183.
- Wu, Z.L. et al. (2017) Glycobiology 27:518.
Citations for Recombinant Human Active Heparanase/HPSE 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.
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Citations: Showing 1 - 10
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A Screening Approach to Assess the Impact of Various Commercial Sources of Crude Marine ?-Carrageenan on the Production of Oligosaccharides with Anti-heparanase and Anti-migratory Activities
Authors: Manseur, C;Groult, H;Porta, M;Bodet, PE;Mersni-Achour, R;Petit, R;Ali-Moussa, S;Musnier, B;Le Cerf, D;Varacavoudin, T;Haddad, O;Sutton, A;Leal, CEY;Alencar-Filho, EB;Piot, JM;Bridiau, N;Maugard, T;Fruitier-Arnaudin, I;
Marine drugs
Species: N/A
Sample Types: Proteins
Applications: Bioassay -
Chemoenzymatic synthesis of sulfur-linked sugar polymers as heparanase inhibitors
Authors: P He, X Zhang, K Xia, DE Green, S Baytas, Y Xu, T Pham, J Liu, F Zhang, A Almond, RJ Linhardt, PL DeAngelis
Nature Communications, 2022-12-02;13(1):7438.
Applications: Bioassay -
A Marine lambda-Oligocarrageenan Inhibits Migratory and Invasive Ability of MDA-MB-231 Human Breast Cancer Cells through Actions on Heparanase Metabolism and MMP-14/MMP-2 Axis
Authors: R Cousin, H Groult, C Manseur, R Ferru-Clém, M Gani, R Havret, C Toucheteau, G Prunier, B Colin, F Morel, JM Piot, I Lanneluc, K Baranger, T Maugard, I Fruitier-A
Marine Drugs, 2021-09-28;19(10):.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
COVID-19 generates hyaluronan fragments that directly induce endothelial barrier dysfunction
Authors: KA Queisser, RA Mellema, EA Middleton, I Portier, BK Manne, F Denorme, EJ Beswick, MT Rondina, RA Campbell, AC Petrey
JCI Insight, 2021-09-08;0(0):.
Species: Human
Sample Types: Plasma
Applications: Bioassay -
Perlecan in the Natural and Cell Therapy Repair of Human Adult Articular Cartilage: Can Modifications in This Proteoglycan Be a Novel Therapeutic Approach?
Authors: J Garcia, HS McCarthy, JH Kuiper, J Melrose, S Roberts
Biomolecules, 2021-01-13;11(1):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure
Authors: K Ko, T Suzuki, R Ishikawa, N Hattori, R Ito, K Umehara, T Furihata, N Dohmae, RJ Linhardt, K Igarashi, T Toida, K Higashi
J Biol Chem, 2020-10-30;0(0):.
Species: Human
Sample Types: Reference Standard
Applications: Bioassay -
Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice
Authors: F Njau, N Shushakova, H Schenk, VC Wulfmeyer, R Bollin, J Menne, H Haller
PLoS ONE, 2020-01-14;15(1):e0218494.
Species: Human
Sample Types: Whole Cells
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Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling
Authors: Y Kiyan, S Tkachuk, K Kurselis, N Shushakova, K Stahl, D Dawodu, R Kiyan, B Chichkov, H Haller
Sci Rep, 2019-09-19;9(1):13591.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
Authors: Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R
Glycobiology, 2018-02-01;0(0):.
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Click Chemistry -
Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of ?-Carrageenan Derivative as a Potent Anti Angiogenic Agent
Authors: N Poupard, P Badarou, F Fasani, H Groult, N Bridiau, F Sannier, S Bordenave-, C Kieda, JM Piot, C Grillon, I Fruitier-A, T Maugard
Mar Drugs, 2017-05-09;15(5):.
Applications: Bioassay -
Production of heparin and ?-carrageenan anti-heparanase derivatives using a combination of physicochemical depolymerization and glycol splitting
Authors: N Poupard, H Groult, J Bodin, N Bridiau, S Bordenave-, F Sannier, JM Piot, I Fruitier-A, T Maugard
Carbohydr Polym, 2017-02-16;166(0):156-165.
Applications: Bioassay -
Heparanase Activates Antithrombin through the Binding to Its Heparin Binding Site
Authors: Nataliya Bohdan
PLoS ONE, 2016-06-20;11(6):e0157834.
Species: Human
Sample Types: Whole Cells
Applications: Western Blot -
Syndecan-3 and TFPI colocalize on the surface of endothelial-, smooth muscle-, and cancer cells.
Authors: Tinholt M, Stavik B, Louch W, Carlson C, Sletten M, Ruf W, Skretting G, Sandset P, Iversen N
PLoS ONE, 2015-01-24;10(1):e0117404.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
FAQs
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What is the best practice for storage of 7570-GH at -20 oC to -70 oC?
7570-GH should be stored at -20 oC to -70 oC undiluted in the vial received or undiluted as 5 µL aliquots. 7570-GH could also be diluted 5-fold in the formulation buffer (specified on the Certificate of Analysis) and stored diluted as 5 µL or larger aliquots at -20 oC to -70 oC.
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