Product Class: Other

Endo H
NEBU cloned at NEB recombinant 37 75 Heat

New Reaction Buffer

Product Introduction

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

  • Recombinant enzyme with no detectable exoglycosidase or other endoglycosidase contaminating activities
  • Glycerol-free for optimal performance in HPLC and mass spectrometry analysis
  • ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
  • Optimal activity and stability for up to 24 months
Catalog # Size Concentration
P0702S 10000.0 units 500000 units/ml
P0702L 50000.0 units 500000 units/ml

Product Information

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 µg 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.

Product Source

Cloned from Streptomyces picatus (2) and overexpressed in E.coli (3).
This product is related to the following categories:
Endoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
Expression Systems,
Glycan Sequencing,
Proteomics,
Recombinant Glycoprotein Expression,
Glycoprotein Analysis

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • P0702S     -20    
      Endo H P0702SVIAL -20 1 x 0.02 ml 500,000 units/ml
      GlycoBuffer 3 B1720SVIAL -20 1 x 1 ml 10 X
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X
  • P0702L     -20    
      Endo H P0702LVIAL -20 1 x 0.1 ml 500,000 units/ml
      GlycoBuffer 3 B1720SVIAL -20 1 x 1 ml 10 X
      Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X

Properties & 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 Buffer

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

Heat Inactivation

75°C for 10 minutes

Molecular Weight

Apparent: 29 kDa

Application Features

  • Removal of high mannose N-glycans from glycoproteins

Product 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.

Protocols, Manuals & Usage

Protocols

  1. Endo H/Endo Hf Protocol
  2. Protocol for Endo H/Hf Non-Denaturing P0702 and P0703

Tools & Resources

Selection Charts

FAQs & Troubleshooting

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

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

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]

Certificate Of Analysis

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

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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