α1-6 Mannosidase

Description

Substrate Specificity:
α1-6 Mannosidase is a highly specific exoglycosidase that removes unbranched α1-6 linked D­­­-mannopyranosyl residues from oligosaccharides (1,2). When used in conjunction with α1-2,3 Mannosidase, the α1-6 Mannosidase will cleave α1-6 Mannose residues from branched carbohydrate substrates.


Figure 1: Detailed specificity of α1-6 Mannosidase
Figure 1: Detailed specificity of α1-6 Mannosidase
All reactions contained 32 units of α1-2,3 Mannosidase (NEB #P0729 ), 40 units of α1-6 Mannosidase, 1X GlycoBuffer 1 and 1X BSA In a total reaction volume of 10 µl. Reactions were incubated at 37°C. The substrate depicted in (E) will not cut to completion. If this structure exists in any substrate it will be impervious to cleavage by α1-6 Mannosidase. Note: When used alone, α1-6 Mannosidase will still act only on linear substrates. When used in conjunction with α1-2,3 Mannosidase, the α1-6 Mannosidase will cleave α1-6 Mannose residues from branched carbohydrate substrates.

Product Source

Cloned from Xanthomonas manihotis and expressed in E. coli (2).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
GlycoBuffer 1-2010X
Purified BSA-20100X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave > 95% of the terminal α-D-mannose from 1 nmol of Manα1-6Manα1-6Man-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37°C in a total reaction volume of 10 µl.

Unit Definition Assay: 
Two fold dilutions of α1­­­­­­­­‑6 Mannosidase are incubated with 1 nmol AMC-labeled substrate in 1X GlycoBuffer 1, supplemented with 100 µg/ml BSA, in a 10 µl reaction. The reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized via thin layer chromatography (1).

Reaction Conditions

1X GlycoBuffer 1
Supplement with 100 μg/ml Purified BSA
Incubate at 37°C

1X GlycoBuffer 1:
5 mM CaCl2
50 mM sodium acetate
pH 5.5 @ 25°C

Storage Temperature

4°C

Storage Conditions

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

Heat Inactivation

65°C for 10 min

Molecular Weight

Apparent: 51 kDa

Notes

  1. p-nitrophenyl-α-D­­­-mannopyranoside is NOT a substrate for this enzyme.
  2. Avoid repeated freeze-thaw cycles.

References

  1. Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.
  2. Guthrie, E.P. and Taron, C.H. New England Biolabs, unpublished observations.

FAQs

  1. What is a good positive control for α1-6 Mannosidase?
  2. How much exoglycosidase should be used?
  3. 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?
  4. Do detergents inhibit exoglycosidases/endoglycosidases?
  5. What are glycosidases and their uses?

Tech Tips

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

When used alone α1-6Man removes only unbranched α1-6 linked mannose residues from oligosaccharides.

p-nitrophenyl-a-D-Mannopyranoside (pNP-Man) is NOT a substrate for this enzyme.

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

Protocols

  1. Typical Reaction Conditions for α1-6 Mannosidase (P0727)

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Usage Guidelines & Tips

Interactive Tools

Quality Control

Quality Assurance Statement

  • No contaminating exoglycosidase or proteolytic activity could be detected (ND).

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Glycosidase Activity (TLC):
    The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide.  The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
  • Protease Activity (SDS-PAGE):
    The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

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]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.