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Endo-H

Endo H cleaves asparagine-linked hybrid or high mannose oligosaccharides but not complex oligosaccharides

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Specsheet   CoA   MSDS

Part Number – Amount of Enzyme
E-EH02     – 60 µLs¹
E-EH02-20   – 20 µls¹
E-EH02-200 – 200 µls²
  ¹ includes buffer and denaturant
  ² includes enzyme only

$180.00$1,344.00

  • 20 µls
  • 60 µls
  • 200 µls
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Endo H, endo-beta-N-acetylglucosaminidase H, Endoglycosidase H

Endo H cleaves Asparagine-linked hybrid or high mannose oligosaccharides, but not complex oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact. Detergent and heat denaturation may increase the rate of cleavage for some glycoproteins.

Source recombinant from Streptomyces plicatus in E.Coli

EC 3.2.1.96

Specific Activity One unit of Endo H activity is defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 µmole of denatured Ribonuclease B. Cleavage is monitored by SDS-PAGE (cleaved Ribonuclease B migrates faster).

Contents
60 µl aliquot of enzyme (300 mU) in 20 mM Tris-HCl, 25 mM NaCl, 1 mM EDTA (pH 7.5).
Included with 20 µL and 60 µL pack sizes:
200 µl 5x Reaction Buffer 5.5 (250 mM sodium phosphate, pH 5.5)
Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanol

Specific Activity >40 U/mg

Activity >5 U/ml

Molecular weight 29,000 daltons

pH range 5-6, optimum 5.5

Suggested usage
1. Add up to 200 µg of glycoprotein to Eppendorf tube. Adjust to 37.5 µl final volume with deionized water.
2. Add 10 µl 5X Endo H Buffer and 2.5 µl of Denaturation Solution (SDS/-ME). Heat at 100˚C for 5 minutes.
3. Cool, and then add 2.0 µl of Endo H to the reaction. Incubate 3 hours at 37˚C.

Storage Store enzyme at 4˚C.

Stability Stable at least 12 months when stored properly. Several days exposure to ambient tempertures will not reduce activity.

Purity Endoglycosidase H is tested for contaminating protease as follows; 10 μg of denatured BSA is incubated for 24 hours at 37oC with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.

The production host strain has been extensively tested and does not produce any detectable glycosidases.

Endo H References

Robbins P. W., D. F. Wirth and C. J. Hering. Expression of the Streptomyces enzyme endoglycosidase H in Escherichia coli. Biol Chem 256:10640-10644 (1981).

Robbins P. W., R. B. Trimble, D. F. Wirth, C. Hering, F. Maley , G. F. Maley, R. Das, B. W. Gibson, N. Royal and K. Biemann. Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H. J Biol Chem 259:7577-7583 (1984).

Trimble R. B., A. L. Tarentino , G. E Aumick and F. Maley. Endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus. Methods Enzymol 83:603-610 (1982).

Trimble R. B. and F. Maley. Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H. Anal Biochem 141:515-522 (1984).

Trimble R. B., R. J. Trumbly and F. Maley. Endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus. Methods Enzymol 138:763-770 (1987).

Trumbly R. J., P. W. Robbins, M. Belfort, F. D. Ziegler, F. Maley and R. B. Trimble. Amplified expression of Streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product. J Biol Chem 260:5683-5690 (1985).

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