Product Class: Other

Protein Deglycosylation Mix II
cloned at NEB recombinant 65 Heat

Product Introduction

The Protein Deglycosylation Mix II contains all of the enzymes, reagents, and controls, to remove all N-linked, as well as many common O-linked glycans, from glycoproteins. Following the deglycosylation reaction, samples are ready to be prepared for mass spectrometry analysis

  • Fast reaction setup
  • Complete sugar removal with no protein degradation
  • Recombinant enzymes with no contaminating endoglycosidase or exoglycosidases
  • Each enzyme exhibits ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
  • All reaction components are compatible with HPLC and mass spectrometry analysis
Catalog # Size Concentration
P6044S 20.0 reactions

Product Information

Description

Glycosylation is one of the most common post-translational modifications of proteins, as shown in Figure 1. N-linked glycosylation occurs when glycans are attached to asparagine residues on the core protein. O-linked glycosylation occurs when glycans are attached to serine or threonine residues. Both chemical and enzymatic methods exist for removing oligosaccharides from glycoproteins. However, chemical methods such as β-elimination with mild alkali or mild hydrazinolysis can be harsh and may result in incomplete sugar removal and degradation of the protein; whereas, enzymatic methods are much gentler and can provide complete sugar removal with no protein degradation.

PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from glycoproteins. PNGase F digestion deaminates the aspargine residue to aspartic acid, and leaves the oligosaccharide intact, keeping it suitable for further analysis. Oligosaccharides containing a fucose α(1-3)-linked to the glycan core are, however, resistant to PNGase F which can occur on some plant and insect glycoproteins.

To remove O-linked glycans, monosaccharides must be removed by a series of exoglycosidases until only the Galβ1-3GalNAc (core 1) and/or the GlcNAcβ1-3GalNAc (core 3) cores remain attached to the serine or threonine. NEB’s O-Glycosidase, cloned from Enterococcus faecalis, can then remove these core structures with no modification of the serine or threonine residues. Any modification of the core structures, including sialyation, will block the action of the O-Glycosidase. Sialic acid residues are easily removed by a general α2-3,6,8,9 Neuraminidase A. In addition, exoglycosidases such as β(1-4)Galactosidase S and β-N-Acetylhexosaminidasef can be included in deglycosylation reactions to remove other complex modifications often known to be present on the core structures. This combination of enzymes may not remove all O-linked oligosaccharides but should remove many common oligosaccharide structures.

The Protein Deglycosylation Mix II contains all of the enzymes, reagents, and controls needed to remove all N-linked and simple O-linked glycans as well as some complex O-linked glycans. This mix contains enzyme sufficient for 20 reactions or the cleavage of as much as 2 mg of glycoprotein. All of the enzymes and reagents included in the Protein Deglycosyation Mix II are Mass Spectrometry compatible. Following the deglycosylation reaction, samples are ready to be prepared for mass spectrometry analysis.

Figure 1: A Glycoprotein modified with O-linked and N-linked glycosylation.



P6044_figure_1


Figure 2




Enzymatic Deglycosylation of Bovine Fetuin under both native (10X Deglycosylation Mix Buffer 1) and reducing (10X Deglycosylation Mix Buffer 2) conditions. 20 µg reactions were loaded onto a 10-20% Tris-glycine SDS-PAGE gel.
Lane 1:    Color Prestained Protein Standard, Broad Range (11-245 kDa) (NEB #P7712)
Lane 2:    20 μg untreated Fetuin control
Lane 3:    20 µg Fetuin deglycosylated under native conditions with Deglycosylation Mix Buffer 1
Lane 4:    20 µg Fetuin deglycosylated under reducing conditions with Deglycosylation Mix Buffer 2
Lane 5:    5 µl Protein Deglycosylation Mix II.

Protein Deglycosylation Mix II:
PNGase F (Glycerol-free), Recombinant:
10,000 units/vial

O-Glycosidase:
800,000 units/vial

α2-3,6,8,9 Neuraminidase A:
400 units/vial

β1-4 Galactosidase S:
960 units/vial

β-N-acetylhexosaminidasef:
300 units/vial

Substrate Control:
Fetuin, 0.5 mg (Fetuin contains sialylated N-linked and O-linked glycans)

Description of Enzymes Included in the Protein Deglycosylation Mix II
O-Glycosidase (NEB #P0733), also known as Endo-α-N-Acetylgalactosaminidase, is a recombinant enzyme cloned from Enterococcus faecalis (1). It catalyzes the removal of core 1 and core 3 O-linked disaccharides from glycoproteins. The molecular weight is approximately 147 kDa.

PNGase F (Glycerol-free), Recombinant (NEB #P0709), also known as Peptide: N-glycosidase F, is cloned from Elizabethkingia miricola (formerly Flavobacterium meningosepticum) and expressed in E. coli (2). PNGase F (Glycerol-free), Recombinant is an amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins unless α(1-3) core fucosylated. The molecular weight is approximately 36 kDa.

α2-3,6,8,9 Neuraminidase A (NEB #P0722), also known as Sialidase A, is a recombinant enzyme cloned from Arthrobacter ureafaciens and expressed in E. coli (3). It catalyzes the hydrolysis of α2,3, α2,6, α2,8 and α2,9 linked N-acetylneuraminic acid residues from glycoproteins and oligosaccharides. The molecular weight is approximately 100 kDa.

β1-4 Galactosidase S (NEB #P0745), is a recombinant enzyme cloned from Streptococcus pneumoniae and expressed in E. coli (4). It is a highly specific exoglycosidase that catalyzes the hydrolysis of β1-4 linked galactose residues from oligosaccharides. The molecular weight is approximately 231 kDa.

β-N-Acetylhexosaminidasef (NEB# P0721), is a recombinant enzyme cloned from Streptomyces plicatus (5) and overexpressed in E. coli (6). It catalyzes the hydrolysis of terminal β-N-acetylgalactosamine and glucosamine residues from oligosaccharides. The molecular weight is approximately 100 kDa.
This product is related to the following categories:
Exoglycosidases Products,
Endoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
Expression Systems,
Protein Digestion,
Glycan Sequencing,
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
  • P6044S     -20    
      Protein Deglycosylation Mix II P6044SVIAL -20 1 x 0.1 ml 20 reactions
      Deglycosylation Mix Buffer 1 B6044SVIAL -20 1 x 1 ml 10 X
      Deglycosylation Mix Buffer 2 B6045SVIAL -20 1 x 1 ml 10 X
      Fetuin P6042SVIAL -20 1 x 0.05 ml 10 mg/ml

Properties & Usage

Storage Buffer

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

Heat Inactivation

65°C for 20 minutes

Application Features

The Protein Deglycosylation Mix II contains all of the enzymes, reagents, and controls needed to remove all N-linked and simple O-linked glycans as well as some complex O-linked glycans. This mix contains enzyme sufficient for 20 reactions or the cleavage of as much as 2 mg of glycoprotein. All of the enzymes and reagents included in the Protein Deglycosyation Mix II are Mass Spectrometry compatible.

Product Notes

  1. The Protein Deglycosylation Mix II is supplied with two different reaction buffers. Please reference the “Protocols and Manuals” tab for the specific protocol associated with each buffer system. The 10X Deglycosylation Mix Buffer 1 (NEB #B6044) should be used when native (non-denaturing) conditions are necessary. The 10X Deglycosylation Mix Buffer 2 (NEB #B6045) will reduce the glycoprotein, but will also provide the most efficient and complete level of deglycosylation. If non-denaturing conditions are not required, we recommend using 10X Deglycosylation Mix Buffer 2.
  2. The Protein Deglycosylation Mix II is not recommended for use on Mucin-like substrates.

References

  1. Koutsioulis, D. Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
  2. Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.
  3. McLeod, E. New England Biolabs, Inc. Unpublished observation
  4. Chen, M. New England Biolabs, Inc. Unpublished observation
  5. Robbins, P. et al. (1992). Gene. 111, 69-76.
  6. Guan, C. and Wong, S. New England Biolabs, Inc. Unpublished observation

Protocols, Manuals & Usage

Protocols

  1. Reaction Protocols for Protein Deglycosylation Mix II (P6044)

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the difference between the discontinued Protein Deglycosylation Mix (NEB# P6039) and the new Protein Deglycosylation Mix II (NEB# P6044)?
  2. Two different reaction buffers (10X Deglycosylation Mix Buffer 1 and 2) are supplied with the Protein Deglycosylation Mix II. How do I know which buffer to use?
  3. Are downstream analysis such as HPLC and Mass Spectrometry compatible with the Protein Deglycosylation Mix II?
  4. I tried the Protein Deglycosylation Mix II on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
  5. How much Protein Deglycosylation Mix II should I use to remove the carbohydrates under native (non-denaturing) conditions?
  6. What is a good control substrate for the Protein Deglycosylation Mix II?
  7. Are Protease Inhibitors acceptable for use with the Protein Deglycosylation Mix II reaction?

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 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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