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  • α1-2,3 Mannosidase

    Description

    Substrate Specificity:
    α1-2,3 Mannosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of α1-2 and α1-3 linked D-Mannopyranosyl residues from oligosaccharides (1).

    Detailed Specificity: Specificity can vary depending on incubation time and concentration of substrate (Fig. 1).

    Figure 1: Detailed specificity of a1,2-3 Mannosidase. All reactions contained 32 units of a1,2-3 Mannosidase, 1X G6 Reaction Buffer and 1X BSA in a total reaction volume of 10 μl. Reactions were incubated at 37°C.

    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
    G6 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 non-reducing terminal α-D-mannose from 1 nmol Manα1-3Manβ1-4GlcNAc-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 serial dilutions of α1-2,3 Mannosidase are incubated with 1 nmol AMC-labeled substrate in 1X G6 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 G6 Reaction Buffer
    Supplement with 5 mM CaCl2
    Incubate at 37°C

    1X G6 Reaction Buffer:
    5 mM CaCl2
    50 mM sodium acetate
    pH 5.5 @ 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: 90 kDa

    Quality Control

    Quality Assurance Statement

    • No contaminating exoglycosidase or proteolytic activity could be detected.

    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.

    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 observation

    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. What is a good positive control for this α1-2, 3 Mannosidase?
    2. How much exoglycosidase should be used?
    3. Do detergents inhibit exoglycosidases/endoglycosidases?
    4. What are glycosidases and their uses?
    1. Typical Reaction Conditions for  α1-2,3 Mannosidase (P0729)

    Usage Guidelines & Tips

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

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

    p-nitrophenyl-a-D-mannopyranoside is NOT a substrate for this enzyme.