Endo H

Description


 
Endoglycosidase H is a recombinant glycosidase which cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins.


60 mg of RNase B was incubated with 3,000 units of Endo H or Endo Hf under standard assay conditions
60 mg of RNase B was incubated with 3,000 units of Endo H or Endo Hf under standard assay conditions.
Aliquots were removed at various time points and measured for released carbohydrate. [Dubois et al. (1956) Anal. Chem. 28, 350-356].
Mobility Shift Analysis
Mobility Shift Analysis
1 unit of Endo H, Endo Hf or PNGase F was incubated per 10 µg of RNase B under standard assay conditions. At various time points, aliquots were removed and analyzed on a 10-20% SDS-PAGE gel for carbohydrate (CHO) release. 1 unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10 µg of RNase B in one hour at 37°C.

Highlights


  • Isolated from a recombinant source

Product Source

Cloned from Streptomyces picatus (2) and overexpressed in E.coli (3).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Glycoprotein Denaturing Buffer-2010X
Glyco Buffer 3-2010X

Advantages and Features

Applications

  • Removal of high mannose N-glycans from glycoproteins

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 µl (10 NEB units = 1 IUB milliunit). 

Unit Definition Assay: 
10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of 1X GlycoBuffer 3, two-fold dilutions of Endo H are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT

Reaction Conditions

1X GlycoBuffer 3 
Incubate at 37°C

1X GlycoBuffer 3:
50 mM sodium acetate
pH 6 @ 25°C

Storage Temperature

-20°C

Storage Conditions

20 mM Tris-HCl
50 mM NaCl
5 mM EDTA
pH 7.5 @ 25°C

Heat Inactivation

75°C for 10 min

Molecular Weight

Apparent: 29 kDa

Notes

  1. Enzymatic activity is not affected by SDS.
  2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
  3. Activity at different temperatures (measured after a 1 hour incubation of glycosidase and denatured RNase B at the given temperature): 37°C - 100%; 30°C - 65%; 25°C - 40%; 17°C - 25% and  2°C - 0%.
  4. Typical reaction conditions: Please see FAQs.

References

  1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
  2. Robbins, P. et al. (1984). J. Biol. Chem. 259, 7577-7583.
  3. Guan, C. and Wong,S. New England Biolabs, unpublished observations.

FAQs

  1. What is the difference between PNGase F, Endo H and O-Glycosidase?
  2. What is the difference between Endo H and Endo Hf?
  3. I tried the Endo H/Hf on my glycoprotein and it failed. What could be the problem?
  4. How much Endo H/Endo Hf should I use?
  5. Is EndoH/ Endo Hf inhibited by SDS?
  6. What are the typical reaction conditions for Endo H?
  7. Are Protease Inhibitors acceptable for use in an Endo H/Hf reaction?
  8. What are glycosidases and their uses?
  9. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
  10. What is a good endoglycosidase substrate?
  11. Do detergents inhibit exoglycosidases/endoglycosidases?

Tech Tips

You can use this enzyme under native or denaturing conditions
Under native conditions we recommend adding more enzyme and using longer incubation times
Enzymatic activity is not affected by SDS
A good positive control substrate is RNase B

Protocols

  1. Endo H/Endo HProtocol

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Feature Articles

Interactive Tools

Citations

  • Rosenbaum EE, Vasiljevic E, Brehm KS, Colley NJ (2014). Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster PLoS Genet. 10(5), e1004349. PubMedID: 24785692, DOI: 10.1371/journal.pgen.1004349
  • Arakel EC, Brandenburg S, Uchida K, Zhang H, Lin YW, Kohl T, Schrul B, Sulkin MS, Efimov IR, Nichols CG, Lehnart SE, Schwappach B (2014). Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes J Cell Sci. 127(Pt 9), 2106-19. PubMedID: 24569881, DOI: 10.1242/jcs.141440
  • Möykkynen T, Coleman SK, Semenov A, Keinänen K (2014). The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization J Biol Chem. 289(19), 13197-205. PubMedID: 24652293, DOI: 10.1074/jbc.M113.526301

Quality Control

Quality Assurance Statement

  • No contaminating endoglycosidase, exoglycosidase or proteolytic activity could be detected.

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Glycosidase Activity (TLC):
    The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
  • Protease Activity (SDS-PAGE):
    The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.