Endo D

Product Information

 
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

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
  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: 140 kDa
  

  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.

• Related Products

  Companion Products

  • Chitin Magnetic Beads
  • 6-Tube Magnetic Separation Rack
  • 12-Tube Magnetic Separation Rack
  • Endo S
  • Remove-iT® PNGase F
  • DTT
  • GlycoBuffer 2

• 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

  • Endoglycosidase Selection Chart

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?

Citations & Technical Literature

• Citations

  Product Citation Tool

• 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]

  • P0742S_L_v2

• 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]

  • P0742S_L_v1_0011606
  • P0742S_L_v1_0011603
  • P0742S_L_v1_0011612
  • P0742S_L_v1_0011706
  • P0742S_L_v1_0011804
  • P0742L_v2_10039713
  • P0742S_v2_10039419
  • P0742L_v2_10053304
  • P0742S_v2_10053306
  • P0742S_v2_10068257
  • P0742L_v2_10065141
  • P0742L_v2_10080275
  • P0742L_v2_10084156
  • P0742L_v2_10093423
  • P0742S_v2_10084153
  • P0742S_v2_10107166
  • P0742L_v2_10096503
  • P0742S_v2_10116016
  • P0742L_v2_10116017
  • P0742L_v2_10143868
  • P0742S_v2_10143865
  • P0742L_v2_10151217
  • P0742L_v2_10167450
  • P0742S_v2_10167451
  • P0742S_v2_10191225
  • P0742S_v2_10223135
  • P0742L_v2_10191224
  • P0742S_v2_10234752
  • P0742L_v2_10235980
  • P0742L_v2_10252797
  • P0742S_v2_10254370

• 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 D
  • DTT
  • GlycoBuffer 2

• 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.

  Trademarks

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