Heparinase 2

Manufactured by New England Biolabs

Sourced in Germany, United Kingdom

Heparinase II is an enzyme used for the digestion and depolymerization of heparin and heparan sulfate. It is a useful tool for the analysis and structural characterization of these glycosaminoglycans.

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Lab products found in correlation

Heparinase 3, by New England Biolabs (2 mentions) Advanced rpmi 1640, by Thermo Fisher Scientific (1 mentions) Dialyzed fbs, by Thermo Fisher Scientific (1 mentions) Proteinase k, by New England Biolabs (1 mentions) Primocin, by InvivoGen (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Tinzaparin, by Leo pharma (1 mentions) Chondroitinase abc chabc, by Merck Group (1 mentions) Heparinase 1, by New England Biolabs (1 mentions) Proteinase k, by Qiagen (1 mentions) Proteinase k, by Merck Group (1 mentions) Amicon ultra 4 centrifugal filter, by Merck Group (1 mentions)

Spelling variants of this product

Heparinase (3 citations) Heparinases I–II–III (2 citations)

6 protocols using heparinase 2

Sulfation Inhibition and Cell Surface Modification

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For sulfation inhibition, cells were grown for 48 h (or other time period if specified) in sulfate-free/NaClO₃-containing media, which consisted of custom-made Advanced RPMI-1640 (Life Technologies) which had 407 μM magnesium sulfate replaced with 407 μM magnesium chloride, 3.03 μM zinc sulfate replaced with 3.03 μM zinc chloride, 5 nM copper (II) sulfate replaced with 5 nM copper (II) chloride, and sodium chloride reduced from 103 mM to 53 mM, and was supplemented with 10% dialyzed FBS (Gibco), 2 mM L-glutamine (Gibco), 100 μg/mL primocin (In vivogen), and 50 mM sodium chlorate (NaClO₃; Sigma-Aldrich) or other NaClO₃ concentration if specified, which was balanced with sodium chloride (Sigma-Aldrich).
For surface enzymatic treatment of cells, 2.5 × 10⁵ cells were washed three times with PBS, and then incubated in PBS containing 2 U/mL heparinase II (New England BioLabs), 1 U/mL proteinase K (New England BioLabs), or no enzyme for 1 h at 37°C/5% CO₂. Cells were then washed in ice-cold PBS and stained as indicated.

Klein K., Hölzemer A., Wang T., Kim T.E., Dugan H.L., Jost S., Altfeld M, & Garcia-Beltran W.F. (2021). A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors. Frontiers in Immunology, 12, 798235.

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Heparinase Pretreatment Enhances ORF2 Binding

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LMH cells were pre-treated in Heparinase Reaction Buffer (20 mM Tris-HCl, 100 mM NaCl and 1.5 mM CaCl₂) with heparinase II (10U/ml) from Bacteroides (New England Biolabs GmbH, Frankfurt am Main, Germany) or PBS as control for 2h at 37°C in an atmosphere supplied with 5% CO₂. After 2h of incubation cells were washed with PBS and incubated with ORF2-3 or ORF2-4 (500nM each) for 1h at 37°C with 5% CO₂. Samples were analyzed by immunofluorescence staining as described for the experiments with heparin sodium salt.

Zhang X., Bilic I., Marek A., Glösmann M, & Hess M. (2016). C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells. PLoS ONE, 11(4), e0153723.

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Heparin-AT Interaction Kinetics

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Human antithrombin (AT) was purchased from Haematologic Technologies (Essex Junction, VT), and heparinase II (2000 U /mL) from Bacteroides eggerthii was purchased from New England Biolabs (Ipswich, MA). Fixed-length heparin oligomers were purchased from Iduron (Alderley Edge, UK). The low-molecular weight heparin Tinzaparin^™ (Leo Pharma, Ballerup, Denmark) was provided by Prof. P.L. Dubin (UMass), and the synthetic pentasaccharide (Fondaparinux) was provided by Prof. Robert J. Linhardt (Rensselaer Polytechnic Institute, Troy, NY). All other chemicals and solvents used in this work were of analytical grades or higher.

Niu C., Zhao Y., Bobst C.E., Savinov S.N, & Kaltashov I.A. (2020). Identification of protein recognition elements within heparin chains using enzymatic foot-printing in solution and on-line SEC/MS. Analytical chemistry, 92(11), 7565-7573.

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Proteoglycan Isolation and Enzymatic Characterization

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Proteoglycans were isolated from the CM by precipitation with cationic detergent cetylpyridinium chloride, and the precipitates were dissolved in isopropanol. The polyanionic macromolecules were precipitated by adding sodium acetate-saturated ethanol and washed in ethanol. The extract was dissolved in ammonium acetate and then treated with chondroitinase AC (ChAC, Sigma-Aldrich), chondroitinase B (ChB, Sigma-Aldrich), chondroitinase ABC (ChABC, Sigma-Aldrich), heparinase I (NEB, Ipswich, MA, UK), heparinase II (NEB), or heparinase III (NEB) for further analysis.

Zhu Y., Lam A.K., Shum D.K., Cui D., Zhang J., Yan D.D., Li B., Xu W.W., Lee N.P., Chan K.T., Law S., Tsao S.W, & Cheung A.L. (2021). Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer. Theranostics, 11(6), 2722-2741.

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Heparinase Treatment for GPC4 Quantification

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Samples containing Nluc-GPC4 were treated with Heparinase II (NEB P0735S) and Heparinase III (NEB P0737S) overnight at 37°C, then subject to DEAE pulldown described above, before luciferase activity was assayed. Untreated samples were similarly incubated at 37°C and used as controls for Heparinase treatment for the DEAE pulldown and luciferase assay.

Huang K, & Park S. (2021). Heparan Sulfated Glypican-4 Is Released from Astrocytes by Proteolytic Shedding and GPI-Anchor Cleavage Mechanisms. eNeuro, 8(4), ENEURO.0069-21.2021.

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Quantification of SARS-CoV-2 Spike Protein in EVs

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The levels of SARS-CoV-2 Spike protein in the plasma (1:5 dilution) and EV lysate (50 μg EV protein) were determined using the SAR-CoV-2 (2019-nCoV) Spike protein ELISA Kit according to the manufacturer instructions (SinoBiological). The sensitivity of the kit is 10.28 pg/mL and previous studies have shown that this sensitivity is enough to detect Spike protein in plasma^{13 (link)
14 (link)}. To check the levels of Spike protein in EVs treated with proteinase K, 100μg of Spike protein-positive COVID-19 EVs were treated with and without 50 μg/ml of proteinase K (Exiqon) for 30 min at 37 °C. Then the reaction was inhibited by incubation with 5mM phenylmethylsulfonyl fluoride (PMSF) for 10 min at 37 °C (Mol. Pharmaceutics 2018, 15, 3, 1073–1080). EVs were also treated with and without heparinase II (0.9 mIU/mL, New England Biolabs) for 3 h at 37 °C and then incubated again with fresh enzyme overnight at 37°C ^{15 (link)}. After proteinase K or heparinase treatment, EVs were washed with PBS and concentrated using Amicon Ultra-4 centrifugal filters (10 kDa, Millipore Sigma, USA).

Fisahn J., Hansen U.P, & Lucas W.J. (1992). Reaction kinetic model of a proposed plasma membrane two-cycle H(+)-transport system of Chara corallina.. Proceedings of the National Academy of Sciences of the United States of America, 89(8), 3261-3265.

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