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  • α1-2 Fucosidase

    Description

    Substrate Specificity: substrate specificity
    α1-2 Fucosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of linear α1-2 linked L-fucopyranosyl residues from oligosaccharides (1). In this case, a linear substrate is defined as having no branching on the adjacent residue.
    Figure 1: Detailed Specificity of α1-2 Fucosidase
    Figure 1: Detailed Specificity of α1-2 Fucosidase
    All reactions contained 1X Glycobuffer 1. All reactions contain 1X BSA in a total volume of 10 μl, and all reactions were incubated at 37°C. All reactions contained 20 units of α1-2 fucosidase. Reaction (C) also contained 20 units of α-N-Acetylgalactosaminidase (#P0734). In Reaction (C), the branched α 1-2 fucose is removed in the presence of both enzymes, but not by α1-2 Fucosidase alone.

    Product Source

    Cloned from Xanthomonas manihotis and expressed in E.coli.

    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 α-L-fucose from 1 nmol of Fucα1-2Galβ1-4Glc-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 α1-2 Fucosidase 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

    Theoretical: 70000 daltons

    Notes

    1. Supplement w/100μ/ml BSA. Incubate at 37°C
    2. p-nitrophenyl-α-L-Fucopyranoside is NOT a substrate for this enzyme.
    3. Repeated freeze/thaw cycles may reduce activity. Recommended storage temperature has changed to 4°C.
    4. Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.

    References

    1. Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.

    FAQs

    1. How much exoglycosidase should be used?
    2. 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?
    3. Do detergents inhibit exoglycosidases/endoglycosidases?
    4. What are glycosidases and their uses?

    Protocols

    1. Typical Reaction Conditions for α1-2 Fucosidase (P0724)

    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.

    Interactive Tools

    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.

    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.