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

324717
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      Overview

      Replacement Information

      Key Specifications Table

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      324717-200MIU
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          Plastic ampoule 200 miu
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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.
          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

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          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.