Endo H

Product Information

 
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

Mobility Shift Analysis

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

  The following reagents are supplied with this product:

  NEB https://www.neb.com/en/#	Component Name	Component https://www.neb.com/en/#	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
  

• Advantages and Features

  Application Features

  • Removal of high mannose N-glycans from glycoproteins

• Related Products

  Companion Products

  • Endo H_f
  • PNGase F
  • PNGase F (Glycerol-free)
  • RNase B
  • Remove-iT® PNGase F
  • Endo S
  • Endo D

• 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 H_f Protocol
  2. Protocol for Endo H/H_f Non-Denaturing P0702 and P0703

Tools & Resources

• Selection Charts

  • Endoglycosidase Selection Chart

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 H_f?
  3. I tried the Endo H/H_f on my glycoprotein and it failed. What could be the problem?
  4. How much Endo H/Endo H_f should I use?
  5. Is EndoH/ Endo H_f inhibited by SDS?
  6. What are the typical reaction conditions for Endo H?
  7. Are Protease Inhibitors acceptable for use in an Endo H/H_f 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

Citations & Technical Literature

• Citations

  Product Citation Tool

  Additional Citations

  • 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: https://www.neb.com/en/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: https://www.neb.com/en/10.1074/jbc.M113.526301
  • 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: https://www.neb.com/en/10.1371/journal.pgen.1004349

• Feature Articles

  • Using Glycosidases to Remove Trim or Modify Glycans on Therapeutic Protein

Quality, Safety & Legal

• Quality Control Assays

  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 & Change Notifications

  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]

  • P0702S_L_v1

• 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]

  • P0702S_L_v1_0181603
  • P0702S_L_v1_0181611
  • P0702S_L_v1_0191612
  • P0702S_L_v1_0191708
  • P0702S_L_v1_0191802
  • P0702L_v1_10021981
  • P0702S_v1_10021982
  • P0702L_v1_10040021
  • P0702S_v1_10040019
  • P0702L_v1_10048769
  • P0702S_v1_10040023
  • P0702S_v1_10048770
  • P0702L_v1_10056020
  • P0702S_v1_10062212
  • P0702L_v1_10062668
  • P0702L_v1_10069239
  • P0702S_v1_10077476
  • P0702S_v1_10081123
  • P0702L_v1_10086145
  • P0702S_v1_10086146
  • P0702S_v1_10096798
  • P0702L_v1_10096801
  • P0702S_v1_10120777
  • P0702L_v1_10124276
  • P0702S_v1_10124277
  • P0702S_v1_10151122
  • P0702S_v1_10163719
  • P0702L_v1_10163718
  • P0702L_v1_10181109
  • P0702S_v1_10181432
  • P0702S_v1_10209944
  • P0702L_v1_10209950
  • P0702S_v1_10245971
  • P0702L_v1_10246132
  • P0702L_v1_10280133

• Safety Data Sheets

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

  • Endo H
  • GlycoBuffer 3
  • Glycoprotein Denaturing Buffer

• 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 busdev@neb.com.
  
  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.