Product Class: Kit

N-Glycan Sequencing Kit

This product has been discontinued on 11/23/2020. The components of this kit can be purchased separately:

α2-3,6,8,9 Neuraminidase A (NEB #P0722)
α2-3 Neuraminidase S (NEB #P0743)
β-N-Acetylglucosaminidase S (NEB #P0744)
β1-4 Galactosidase S (NEB #P0745)
α1-3,4,6 Galactosidase (NEB #P0747)
α1-2,4,6 Fucosidase O (NEB #P0749)
α1-2,3,6 Mannosidase (NEB #P0768)

  • Catalog # E0577 was discontinued on November 23, 2020

Product Introduction

The N-Glycan Sequencing Kit consists of seven well characterized, highly pure, recombinant exoglycosidase enzymes selected to simplify the process of characterizing typical N-linked glycan structures.

  • Recombinant enzymes with no detectable endoglycosidase or other exoglycosidase contaminating activities
  • Compatible with N-linked glycans released from a variety of CHO and murine derived antibodies, as well as N-linked glycans released from other glycoproteins
  • Simultaneous digestion with other exoglycosidases
  • Optimal activity and stability for up to 12 months

Storage Temperature: 4°C

Bioz Badge Exists : True
Catalog # Size Concentration

Product Information

Description

The N-Glycan Sequencing Kit consists of seven well characterized, highly pure, recombinant exoglycosidase enzymes selected to simplify the process of characterizing typical N-linked glycan structures. The extreme diversity of glycan structures on a glycoprotein makes elucidation of the profile challenging and often a number of orthogonal approaches are employed to verify the individual structures. The use of sequential or tandem exoglycosidase digestion of oligosaccharides followed by mass spectrometry or capillary electrophoresis analysis provides detailed carbohydrate sequence information and resolves ambiguities. The exoglycosidases are all active in a universal buffer system, GlycoBuffer 1, and can be used in single digests or in combination.

This kit is compatible with N-linked glycans released from a variety of CHO and murine derived antibodies, as well as N-linked glycans released from other glycoproteins. Optimal incubation times and enzyme concentration may vary for more complex glycans. The recommended enzyme quantity is sufficient for digestion of up to 130 pmol of released glycans (equivalent to released glycans from approximately 10 µgs of antibody) labeled with 2AA, 2AB or procainamide. Other commercially available “Instant labels” may require a larger quantity of enzyme or a longer incubation time for complete digestion.

 

Figure 1: Glycan Sample Preparation Workflow

 

 

Figure 2: N-Glycans released from Infliximab, labelled with procainamide, digested for 3 hours with exoglycosidases
 

Digestion of Infliximab with a sequential panel of exoglycosidases serves as a tool to elucidate and verify glycan profiles. Refer to the product manual for reaction conditions.


This product is related to the following categories:

Properties & Usage

Features

  • N-glycan sequencing of antibodies
  • The enzymes provided are all active in a universal buffer system, GlycoBuffer 1, and can be used in single digests or in any combination.
  • All reagents supplied are mass spectrometry compatible.

Protocols, Manuals & Usage

Protocols

  1. Protocols for E0577

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

FAQs & Troubleshooting

FAQs

  1. Is the N-Glycan Sequencing Kit protocol suitable for digestion of glycoproteins?
  2. How do I calculate the molarity of released glycans from my antibody substrate?
  3. Why are two different neuraminidase enzymes provided with the N-Glycan Sequencing Kit?
  4. Should I use the one- or two-step Rapid PNGase F deglycosylation method for my antibody?
  5. The deglycosylation protocol included with the N-Glycan Sequencing Kit is recommended for up to 100 µg of antibody; How do I determine the amount of starting material I should deglycosylate?
  6. Is it possible to combine the 1-236 Mannosidase with osidase actither exoglycosidases Are any of the exoglycosidases inhibited by the Zinc that is required for optimal mannovity
  7. Does ammonium formate inhibit exoglycosidase digestion?
  8. How long do I need to incubate a Rapifluor-labelled substrate with the exoglycosidases?
  9. Which is the enzyme of choice to release α1-6 fucose residues from antibody glycans: α1-2,4,6 Fucosidase O or α1-2,3,4,6 Fucosidase from Bovine Kidney?

Tech Tips

Remove all traces of Acetonitrile before digesting labeled substrate with exoglycosidases for optimal results.

 

Use PCR tubes to avoid evaporation when incubating reactions overnight.

 

Include ZnCl2 in reactions with α1-2,3,6 Mannosidase (P0768) for optimal digestion.

 

Fucosidase O (P0749) requires an 18 hr incubation for complete digest of some instant labelled glycans and complex oligosaccharides with multiple branches.

 

If using Fucosidase O (P0749) alone, incubation temperature can be increased to 50°C for more efficient digestion.

 

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]

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.

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