Product Class: Other

α1-2,3,6 Mannosidase
NEBU cloned at NEB recombinant 37 95 Heat

Product Introduction

α1-2,3,6 Mannosidase is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing α1-2, α1-3 and α1-6 linked mannose residues from oligosaccharides.
• Recombinant enzyme with no detectable endoglycosidase or other exoglycosidase 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
P0768S 80.0 units 2000 units/ml

Product Information

Description

α1-2,3,6 Mannosidase, cloned from Jack Bean, and also known as JBM, is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal α1-2, α1-3 and α1-6 linked mannose residues from oligosaccharides. α1-2,3,6 Mannosidase has a slight preference for α1-2 mannose residues over α1-3 and α1-6 mannose residues.

Substrate Specificity

Product Source

Cloned from Canavalia ensiformis (Jack Bean) and expressed in Pichia pastoris.
This product is related to the following categories:
Exoglycosidases Products,
Proteome Analysis Products,
This product can be used in the following applications:
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
  • P0768S     4    
      α1-2,3,6 Mannosidase P0768SVIAL 4 1 x 0.04 ml 2,000 units/ml
      GlycoBuffer 4 B1703SVIAL -20 1 x 1 ml 10 X
      Zinc B0768SVIAL 4 1 x 1 ml 10 X

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave > 95% of the terminal mannose from 1 nmol of Man(α1,3)-Man(β1,4)-GlcNAc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37°C in a total reaction volume of 10 µl.

Reaction Conditions

1X GlycoBuffer 4
Supplement with 1X Zinc
Incubate at 37°C

1X GlycoBuffer 4
50 mM sodium acetate
(pH 4.5 @ 25°C)

Storage Buffer

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

Heat Inactivation

95°C for 10 minutes

Molecular Weight

Theoretical: 110 kDa

Unit Assay Conditions

Two fold dilutions of α1-2,3,6 Mannosidase are incubated with 1 nmol AMC-labeled substrate, 1X GlycoBuffer 4 and 1X Zinc in a 10 µl reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via thin layer chromatography (1).

Product Notes

  1. α1-2,3,6 Mannosidase is a glycosylated protein. The theoretical molecular weight is 110 kDa, with two subunits at 66 kDa and 44 kDa (2).
  2. The optimal pH for α1-2,3,6 Mannosidase is 4.5. Enzyme activity is enhanced by the presence of 2 mM Zn2+ in the reaction.
  3. In order to achieve complete removal of all non-reducing terminalα-linked mannose residues, it may be necessary to use an increasedenzyme concentration as well as an overnight incubation time (18 hours).
  4. To achieve complete removal of α1-6 mannose residues from a complexsubstrate, a sequential digest using α1-2,3,6 Mannosidase followed byα1-6 Mannosidase (NEB #P0727) may be necessary. 
  5. Reactions may be scaled-up linearly to accommodate larger reaction volumes.
  6. The amount of exoglycosidase enzyme required varies when differentsubstrates are used. Start with 1-2 μl for 1 μg of glycoprotein or 100nM of oligosaccharide for one hour in a 10-25 μl reaction. If there isstill undigested material, let the reaction go overnight.

References

  1. Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.
  2. Kumar, S.N. et al (2014). Glycobiology. 24, 252-261.

Protocols, Manuals & Usage

Protocols

  1. Typical Reaction Conditions for α1-2,3,6 Mannosidase (P0768)

Usage & Guidelines

Application Notes

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the difference between α1-2,3 Mannosidase, α1-6 Mannosidase and α1-2,3,6 Mannosidase? 
  2. Can α1-2,3,6 Mannosidase (JBM) be a substrate itself for mannosidase activity?
  3. What is a good positive control for α1-2,3 Mannosidase?
  4. When digesting complex substrates that include α1-6 Mannose residue, can additional mannose enzymes be used in combination with α1-2,3,6 Mannosidase?
  5. Does this enzyme require denaturing conditions to act on glycoproteins?
  6. What is a good method to re-purify a glycan or glycopeptide after exoglycosidase treatment?
  7. How much exoglycosidase should be used?
  8. Do detergents inhibit glycosidases?
  9. What are Glycosidases and their uses?
  10. How much exoglycosidase should be used?

Tech Tips

Repeated freeze/thaw cycles may reduce activity. Recommended storage temperature is 4°C.

The optimal pH for α1-2,3,6 Mannosidase is 4.5. This differs from the majority of exoglycosidases, which have an optimal pH at 5.5.

Enzyme activity is enhanced by the presence of 2 mM Zn2+ in the reaction.

Using 1-2 µl is a good starting point for a 1 hr incubation of 1 µg of glycoprotein or 100 nM of oligosaccharide.

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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