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  • β-N-Acetylhexosaminidasef

    Description

    β-N-Acetylhexosaminidasef is a recombinant protein fusion of β-N-Acetyl-hexosaminidase (1) and maltose binding protein. It has identical activity to β-N-Acetyl-hexosaminidase. β-N-Acetyl-hexosaminidasef catalyzes the hydrolysis of terminal β-D-N-acetyl-galactosamine and glucosamine residues from oligosaccharides.  β-N-Acetyl-hexosaminidasef can be used to remove O-GlcNAc (N-Acetylglucosaminidase on serine/threonine)(4). 

    Substrate Specificity:

    Highlights

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

    Product Source

    Cloned from Streptomyces plicatus (1) and overexpressed in E. coli (2).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    G2 Reaction Buffer10X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave > 95% of the terminal β-D-N-acetyl-galactosamine from 1 nmol of GalNAcβ1-4Galβ1-4Glc-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 β-N-Acetyl-hexosaminidasef are incubated with 1 nmol AMC-labeled substrate in 1X G2 Reaction Buffer 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 G2 Reaction Buffer
    Incubate at 37°C

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

    Storage Conditions

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

    Heat Inactivation

    75°C for 10 min

    Molecular Weight

    Theoretical: 100000 daltons

    Terminal Monosacaride

    • N-Acetyl-galactosamin and N-Acetyl-glucosamin Residues

    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.

    References

    1. Robbins, P. et al. (1992). Gene. 111, 69-76.
    2. Guan, C. and Wong, S. New England Biolabs. Unpublished observation
    3. Wong-Madden, S.T. and Landry D. (1995). Glycobiology. 5, 19-28.
    4. Magnelli, P.E. et al Methods Molecular Biology. 801, 189-211.

    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. How much exoglycosidase should be used?
    2. Do detergents inhibit exoglycosidases/endoglycosidases?
    3. What is a good positive control for β-N-Acetylhexosaminidasef?
    4. What are glycosidases and their uses?
    1. Typical Reaction Conditions for β-N-Acetylhexosaminidasef (P0721)

    Usage Guidelines & Tips

    ß-N-Acetylhexosaminidasef (P0721)

    A good positive control for ßHexf is pNP-GlcNAc.

    ßHexf 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.