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  • β-N-Acetylglucosaminidase

    Description

    Substrate Specificity

    β-N-Acetylglucosaminidase is a highly specific exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing β-N-Acetylglucosamine residues from oligosaccharides.

    Detailed Specificity
    Specificity can vary depending on incubation time and branching structure.

    Figure 1: Detailed specificity of β-N-Acetylglucosaminidase
     Figure 1: Detailed specificity of β-N-Acetylglucosaminidase
    All reactions contained 4 units of β-N-Acetylglucosaminidase, 1X GlycoBuffer 1 and 1X BSA in a total reaction volume of 10 µl. Reactions (B), (C) and (D) were treated with 8 units of β1-4 Galactosidase prior to treatment with β-N-Acetylglucosaminidase to form the above substrates. Reactions were incubated at 37°C.

    Highlights

    • Epigenetics applications:  O-GlcNAc (N-Acetylglucosamine attached to serine or threonine) is a regulator of nuclear protein function. N-Acetylglucosaminidase removes O-GlcNAc from transcription factors, histones, RNA-binding proteins, etc.

    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
    Purified BSA-2010X
    GlycoBuffer 1-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave > 95% of the terminal, non-reducing β-N-Acetylglucosamine from 1 nmol GlcNAcβ1-4GlcNAcβ1-4GlcNAc-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 β-N-Acetylglucosaminidase 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 (3).

    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

    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: 71000 daltons

    Notes

    1. Recommended storage temperature is 4°C.
    2. Avoid repeated freeze/thaw cycles.

    References

    1. Guthrie, E.P., Shimer, E.P. New England Biolabs, unpublished observations.
    2. Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.
    3. Magnelli, P.E., et al. (2011). Methods Mol. Biol. 801, 189-211.

    FAQs

    1. Can I use β-N-Acetylglucosaminidase in a double digest with other exoglycosidases and/or endoglycosidases?
    2. What is the difference between β-N-Acetylglucosaminidase and β-N-Acetylhexosaminidasef?
    3. Do detergents inhibit β-N-Acetylglucosaminidase?
    4. What is a good positive control for β-N-Acetylglucosaminidase?
    5. What are glycosidases and their uses?
    6. What are the typical reaction conditions for β-N-Acetylglucosaminidase
    7. How much exoglycosidase should be used?
    8. 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?

    Tech 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.

    A good positive control for ßGICNAcase is pNP-GlcNAc                                    

    ß-N-Acetylglucosaminidase is not a general ß-hexosamindase, it is instead a highly specific exoglycosidase that catalyzes the hydrolysis of all linkages of terminal, non-reducing ß-N-acetylglucosamine (ß-GlcNAc) residues from oligosaccharides

    Protocols

    1. Removal of terminal N-acetylglucosamine from the biantennary N-linked sugars of IgG
    2. Typical Reaction Conditions (P0732)

    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

    Application Notes

    NEB Publications

    • Magnelli P, Bielik A, Guthrie E.  (2012). Identification and characterization of protein glycosylation using specific endo- and exoglycosidases Methods Mol Biol. . 801, 189-211. PubMedID: 21987255

    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.