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  • 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. All reactions contained 32 units of α1-2,3 Mannosidase (NEB #P0729 ), 40 units of α1-6 Mannosidase, 1X G6 reaction buffer 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
    G2 Reaction Buffer10X
    BSA-2010 mg/ml

    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 G2 Reaction Buffer, 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 G2 Reaction Buffer
    Incubate at 37°C

    1X G2 Reaction Buffer:
    50 mM sodium citrate
    pH 4.5 @ 25°C

    Storage Temperature

    4°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    1 mM Na2EDTA
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 10 min

    Molecular Weight

    Apparent: 51 kDa

    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.
    • 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.

    Notes

    1. p-nitrophenyl-α-D­­­-mannopyranoside is NOT a substrate for this enzyme.
    2. A double digest with α1-2,3 Mannosidase requires the following reaction conditions: 1X G6 Reaction Buffer: 50 mM Sodium Acetate (pH 5.5 @ 25°C), 5 mM CaCl2. Supplement with 100 µg/ml BSA. Incubate at 37°C.
    3. 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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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.
    1. Do detergents inhibit exoglycosidases/endoglycosidases?
    2. What is a good positive control for α1-6 Mannosidase?
    3. What are Glycosidases and their uses?
    4. How much exoglycosidase should be used?
    1. Typical Reaction Conditions for α1-6 Mannosidase (P0727)

    Usage Guidelines & Tips

    α1-6Man can be used in double digests with other exoglycosidases using G6 Reaction Buffer.

    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.