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  • α1-3, 6 Galactosidase

    Description

     α1-3, 6 Galactosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of α1-3, 6 linked D-galactopyranosyl residues from oligosaccharides.

    Detailed Specificity: Specificity can vary depending on incubation time and branching structure.
    Figure 1: Detailed specificity of α1-3, 6 Galactosidase
    Figure 1: Detailed specificity of α1-3, 6 Galactosidase
    Reaction (B) contained 4 units of α1-3, 6 Galactosidase, 1X GlycoBuffer 1 and 1X BSA in a total reaction volume of 10 µl. Reaction (B) shows that branched fucose inhibits cleavage. Reaction (A) contained 24 units of α1-3, 6 Galactosidase and 100 units of Neuraminidase, followed by a heat kill at 65°C for 10 minutes and a 2 hour digestion with 16 units of β1-4 Galactosidase. The reaction in (B) contained 1X GlycoBuffer 1 and 1X BSA in a total reaction volume of 20 µl. The reactions were incubated at 37°C. Complete digestion of the α1-3, 6 Galactosidase was determined by an observation of complete transformation of the substrate in (A) to the non-reducing terminal N-acetylglucosamine tetra antennary oligosaccharide.

    Highlights

    Removal of the antigenic Galα1,3Gal epitope from glycoproteins.

    Product Source

    Cloned from Xanthomonas manihotis and expressed in E. coli (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    GlycoBuffer 1-2010X
    Purified BSA-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave > 95% of the terminal, α-D-galactose from 1 nmol Galα1-3Galβ1-4Gal-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 α1-3, 6 Galactosidase are incubated with 1 nmol AMC-labeled substrate in 1X GlycoBuffer 1 and 1X 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 (2).

    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: 70000 daltons

    Notes

    1. Avoid repeated freeze-thaw cycles.

    References

    1. McLeod, E. New England Biolabs, Inc., unpublished observations.
    2. Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.

    FAQs

    1. Can I use α1-3,6 Galactosidase in a double digest with other exoglycosidases and/or endoglycosidases?
    2. What is a good positive control for α1-3,6 Galactosidase?
    3. What are glycosidases and their uses?
    4. How much exoglycosidase should be used?
    5. 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?

    Protocols

    1. Protocol for α1-3,6 Galactosidase (P0731)
    2. Typical Reaction Conditions for α1-3, 6 Galactosidase (P0731)

    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

    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.