β1-4 Galactosidase

Description

Substrate Specificity:
β1-4 Galactosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of β1-4 linked D-galactopyranosyl residues from oligosaccharides.

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

Figure 1: Detailed specificity of β1-4 Galactosidase
Figure 1: Detailed specificity of β1-4 Galactosidase
A. 0.1 nm/µl substrate, 2 units, 1 hr incubation
Figure 1: Detailed specificity of β1-4 Galactosidase
B. 0.1 nm/µl substrate, 4 units, 1 hr incubation
Figure 1: Detailed specificity of β1-4 Galactosidase
C. 0.1 nm/µl substrate, 8 units, 1 hr incubation

Reactions (A), (B) and (C) contained 2 units, 4 units and 8 units of β1-4 Galactosidase, respectively, and either 1X GlycoBuffer 1 total reaction volume of 10 µl. Reactions were incubated at 37°C.

Product Source

Cloned from Bacteroides fragilis and expressed in E.coli (1).

Reagents Supplied

The following reagents are supplied with this product:

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

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave > 95% of the terminal, β-D-galactose from 1 nmol Galβ1-4GlcNAcb1-3Galβ1-4Glc-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 serial dilutions of β1-4 Galactosidase are incubated with 1 nmol AMC-labeled substrate in 1X GlycoBuffer 1, 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
Incubate at 37°C

1X GlycoBuffer 1:
5 mM CaCl2
50 mM sodium acetate
pH 5.5 @ 25°C

Storage Temperature

-20°C

Storage Conditions

20 mM Tris-HCl
50 mM NaCl
1 mM EDTA
pH 7.5 @ 25°C

Heat Inactivation

65°C for 10 min

Molecular Weight

Apparent: 94000 daltons

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 McLeod, E. New England Biolabs, Inc., unpublished observations.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 β1-4 Galactosidase in a double digest with other exoglycosidases and/or endoglycosidases?
  2. What is the difference between β1-4 Galactosidase and β1-3 Galactosidase?
  3. What is a good positive control for β1-4 Galactosidase?
  4. What are glycosidases and their uses?
  5. How much exoglycosidase should be used?
  6. Which enzyme should be used to remove β1-4 linked Galactose residues from intact IgG?
  7. 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

ß 1-4 Galactosidase (P0730)

A good positive control for ß 1-4Gal is pNP-ßGal

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

Protocols

  1. Typical Reaction Conditions for β1-4 Galactosidase (P0730)

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. Further information regarding NEB product quality can be found here.
  • 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.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

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.