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324717 Endoglycosidase H, Streptomyces plicatus, Recombinant, E. coli

Endoglycosidase H, Streptomyces plicatus, Recombinant, E. coli cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide.

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Endo-β-N-acetylglucosaminidase H, Endo H

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324717
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      Description
      OverviewRecombinant, streptomyces plicatus endoglycosidase H expressed in E. coli. Catalyzes the hydrolysis of asparagine-linked oligomannose and hybrid, but not complex oligosaccharides from glycoprotein. Endo H cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide.
      Note: 1 mU = 1 milliunit.
      Catalogue Number324717
      Brand Family Calbiochem®
      SynonymsEndo-β-N-acetylglucosaminidase H, Endo H
      References
      ReferencesRao, V., et al. 1995. Structure 3, 449.
      Trimble, R.B., et al. 1987. Methods Enzymol. 138, 763.
      Trumbly, R.J., et al. 1985. J. Biol. Chem. 260, 5683.
      Product Information
      CAS number52769-51-4
      Activity≥3 units/ml
      Unit of DefinitionOne unit is defined as the amount of enzyme that will catalyze the release of N-linked oligosaccharides from 1.0 µmol denatured ribonuclease B per min at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE.
      EC number3.2.1.96
      FormLiquid
      FormulationIn 50 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.5.
      Quality LevelMQ100
      Applications
      Biological Information
      Specific Activity≥30 units/mg protein
      Physicochemical Information
      ContaminantsN-acetylglucosaminidase, α- and β-galactosidase, α-mannosidase, neuraminidases, proteases: none detected
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      324717-200MIU 04055977215984

      Documentation

      Endoglycosidase H, Streptomyces plicatus, Recombinant, E. coli SDS

      Title

      Safety Data Sheet (SDS) 

      Endoglycosidase H, Streptomyces plicatus, Recombinant, E. coli Certificates of Analysis

      TitleLot Number
      324717

      References

      Reference overview
      Rao, V., et al. 1995. Structure 3, 449.
      Trimble, R.B., et al. 1987. Methods Enzymol. 138, 763.
      Trumbly, R.J., et al. 1985. J. Biol. Chem. 260, 5683.

      Citations

      Title
    • Aipo Diao, et al. (2008) Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner. Journal of Biological Chemistry 283, 6957-6967.
      		•
      Data Sheet

      Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

      Revision19-June-2008 RFH
      SynonymsEndo-β-N-acetylglucosaminidase H, Endo H
      DescriptionRecombinant, streptomyces plicatus endoglycosidase H expressed in E. coli. Catalyzes the hydrolysis of asparagine-linked oligomannose and hybrid, but not complex oligosaccharides from glycoprotein. Endo H cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide.
      FormLiquid
      FormulationIn 50 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.5.
      Recommended reaction conditions
      Endoglycosidase H, Streptomyces plicatus, Recombinant, E. coli Note: This protocol is provided only as a general guideline. Researchers should standardize this assay for their own specific needs and should consult published literature. Endoglycosidase H cleaves asparagine-linked oligomannose and hybrid, but not complex, oligosaccharides from glycoproteins. It cleaves between the two Nacetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, N-glycosidase F (Cat. No. 362185) removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins. Required Reagents • Substrate: Ribonuclease B at a concentration of 1.1 mg/ml in 1X assay buffer • Enzyme: Dilute the preparation of Endoglycosidase H 1:400 in 1X assay buffer • 5X Assay Buffer: 250 mM NaHPO4, pH 5.5 • Denaturation buffer: 2% SDS, 1 M β-mercaptoethanol (β-ME) • dH2O • SDS-PAGE system Protocol 1. Add 25 µl of denaturation buffer to 0.45 ml of substrate solution and heat to 100°C for 10 min. Cool and add 25 µl of dH2O. 2. Add 50 µl (50 µg) of denatured substrate to each of 6 tubes. 3. Add 0, 1, 2, 3, 4, or 5 µl of diluted enzyme to appropriately labeled tubes and incubate 1 h at 37°C. 4. Stop the reaction by heating to 100°C for 5 min. 5. Run 10 µl of each sample on a 12% SDS-PAGE gel. Stain with Coomassie Blue. Ribonuclease B is ~15 kD. Note the lowest tube number in which ~50% of the substrate is cleaved. 6. Calculate the activity: Units/ml = (4 X 510) R/M T V R = 25 (µg of Ribonuclease B cleaved) M = 15,000 (formula weight of Ribonuclease B) T = 60 (time in min) V = volume (µl) of enzyme dilution needed to cleave 50% For example, if 2 µl of enzyme resulted in 50% cleavage, the activity would be 5.55 units/ml. Assay for Deglycosylation Required Reagents • Glycoprotein substrate • Endoglycosidase H • 5X Assay Buffer: 250 mM NaHPO4, pH 5.5 • Denaturation buffer: 2% SDS, 1 M β-mercaptoethanol (β-ME) • dH2O • SDS-PAGE system Protocol 1. Add up to 200 µg of glycoprotein to an eppendorf tube. Adjust the final volume to 37.5 µl with dH2O. 2. Add 10 µl of 5X assay buffer and 2.5 µl of denaturation buffer. Heat to 100°C for 5 min. Note: It is not necessary to add Triton® X-100 detergent. SDS will not inactivate the Endoglycosidase H 3. Cool the tube and add 2 µl of Endoglycosidase H to the reaction. Incubate for 3 h at 37°C. Note: If SDS or heat denaturation is omitted, it may be necessary to increase the incubation time. 4. Monitor the cleavage by SDS-PAGE.
      CAS number52769-51-4
      EC number3.2.1.96
      ContaminantsN-acetylglucosaminidase, α- and β-galactosidase, α-mannosidase, neuraminidases, proteases: none detected
      Specific activity≥30 units/mg protein
      Activity≥3 units/ml
      Unit definitionOne unit is defined as the amount of enzyme that will catalyze the release of N-linked oligosaccharides from 1.0 µmol denatured ribonuclease B per min at 37°C, pH 5.5. Cleavage is monitored by SDS-PAGE.
      Storage +2°C to +8°C
      Do Not Freeze Yes
      Toxicity Standard Handling
      ReferencesRao, V., et al. 1995. Structure 3, 449.
      Trimble, R.B., et al. 1987. Methods Enzymol. 138, 763.
      Trumbly, R.J., et al. 1985. J. Biol. Chem. 260, 5683.
      Citation
    • Aipo Diao, et al. (2008) Coordination of golgin tethering and SNARE assembly: GM130 binds syntaxin 5 in a p115-regulated manner. Journal of Biological Chemistry 283, 6957-6967.
      		•