Product Class: Other

Endo D
NEBU cloned at NEB recombinant 37 65 Heat

Product Introduction

Endo D is an endoglycosidase that cleaves within the chitobiose core of paucimannose N-linked glycans, with or without extensions in the antennae, from glycoproteins and glycopeptides.

  • Tagged with a chitin binding domain (CBD) for easy removal
  • Recombinant enzyme with no detectable exooglycosidase 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 12 months
Catalog # Size Concentration
P0742S 1500.0 units 50000 units/ml
P0742L 7500.0 units 50000 units/ml

Product Information

Description


 
Endo D, also known as Endoglycosidase D, is a recombinant glycosidase, which cleaves within the chitobiose core of paucimannose N-linked glycans, with or without extensions in the antennae. Endo D is tagged with a chitin binding domain (CBD) for easy removal from a reaction, and is supplied glycerol-free for optimal performance in HPLC and MS- intensive methods.

Product Source

A truncated Endo D gene cloned from Streptococcus pneumoniae and expressed in E. coli as a fusion to chitin binding domain

Specificity

Detailed Specificity
Figure 1: Detailed specificity of Endo D
Figure 1: Detailed specificity of Remove-iT Endo D
The reaction contained 0.08 mU of α1-2 Mannosidase (Prozyme #GKX-5009), 50 units of Endo D and 2 μl 0.5 M Na0Ac pH 5.0 buffer in a total reaction volume of 10 μl. Reactions were incubated at 37°C for 2 hours. Following removal of the bottom branch, Endo D is active on a complex upper arm (1).
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
  • P0742S     4    
      Endo D P0742SVIAL 4 1 x 0.03 ml 50,000 units/ml
      DTT B0706SVIAL -20 1 x 1 ml 0.4 M
      GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
  • P0742L     4    
      Endo D P0742LVIAL 4 1 x 0.15 ml 50,000 units/ml
      DTT B0706SVIAL -20 1 x 1 ml 0.4 M
      GlycoBuffer 2 B3704SVIAL -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 glycosidase-trimmed (trimannosyl core) Fetuin in 1 hour at 37°C in a total reaction volume of 10 μl.

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

1X GlycoBuffer 2
50 mM Sodium Phosphate
(pH 7.5 @ 25°C)

Storage Buffer

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

Heat Inactivation

65°C for 10 minutes

Molecular Weight

Apparent: 140000 daltons

Unit Assay Conditions

10 μg of glycosidase-trimmed (trimannosyl core) Fetuin are denatured with 1X DTT at 95°C for 3 minutes. After the addition of 1X GlycoBuffer 2, two-fold dilutions of Endo D are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products is visualized by SDS-PAGE.

Product Notes

  1. To deglycosylate a native glycoprotein, longerincubation time, as well as more enzyme, maybe required.
  2. Endo D is not recommended foruse with Glycoprotein Denaturing Buffercontaining both SDS and DTT, as Endo D is inhibited by SDS and, unlike otherendoglycosidases, NP-40 does not counteractthe SDS inhibition.
  3. Removal of Endo D from thedeglycosylation reaction can be scaled uplinearly with larger amounts of chitin magneticbeads.
  4. Chitin Magnetic Beads Binding Capacity is0.4 μg/μl of CBD-tagged protein.
  5. 50 μl of chitin slurry are sufficient to remove 1-5 μl of Endo D (50-250 units)

References

  1. McLeod, E., Shi, S. and Magnelli, P., New England Biolabs, Inc., unpublished results. Unpublished observation

Protocols, Manuals & Usage

Protocols

  1. Reaction Conditions for Endo D (P0742)
  2. Endo D Removal Magnetic Chitin Bead Protocol (P0742)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the tag on Endo D?
  2. What is the difference between PNGase F, Endo S and Endo D?
  3. What is a typical reaction protocol for Endo D?
  4. Will SDS inhibit Endo D?
  5. How do I eliminate Endo D from a reaction?
  6. What is the binding capacity of the Magnetic Chitin Beads used to remove Endo D?
  7. Is Endo D compatible with downstream analysis such as HPLC and Mass Spectrometry?
  8. 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?
  9. Can Endo D cleave glycoproteins under native conditions?

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.

Trademarks

NEW ENGLAND BIOLABS® is a registered trademark of New England Biolabs, Inc.
REMOVE-IT™ is a trademark of New England Biolabs, Inc.

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