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Exoglycosidases >
β1-3 Galactosidase |
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Prices are in US dollars and valid only for US orders.
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Substrate Specificity:
Description:
β1-3 Galactosidase is a highly specific exoglycosidase that catalyzes the hydrolysis of β1-3 and, at a much lower rate, β1-6 linked D-galactopyranosyl residues from oligosaccharides. The approximate kinetic data show > 100-fold preference for β1-3 over β1-6 linkages (1,2), and > 500-fold preference from β1-3 over β1-4 linkages (3).
Detailed Specificity: The GlcNAc(β1-6) residue is the only anomeric configuration that can effect the specificity of the enzyme enabling cleavage of the non-reducing β1-4Galactose (Fig. 1).
Figure 1: Selling concentration of the enzyme will cut the b1-4Galactose linkage as shown in (A) due to the adjacent GlcNAcb1-6 anomer. This cleavage will not occur if the selling concentration of the enzyme is diluted 16-fold, as shown in (B).
Source:
Cloned from Xanthomonas manihotis and expressed in E. coli at NEB (4).
Reagents Supplied:
G2 Reaction Buffer (10X)
BSA (100X)
Enzyme Properties
Molecular Weight:
Theoretical: 66,000 daltons
Reaction & Storage Conditions
Reaction Conditions:
1X G2 Reaction Buffer
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X G2 Reaction Buffer:
50 mM sodium citrate
pH 4.5 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to cleave > 95% of the terminal β-D-galactose from 1 nmol of Galβ1-3GlcNAcβ1-3Galβ1-4Glc-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 dilutions of β1-3 Galactosidase are incubated with 1 nmol AMC-labeled substrate in 1X G2 Buffer, 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).
Concentration:
10,000 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
0.1 mM EDTA
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Avoid repeated freeze/thaw cycles.
Notes
Usage notes:- Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
FAQs
- How much exoglycosidase should be used?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- What are Glycosidases and their uses?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
No contaminating exoglycosidase or proteolytic activity could be detected.
Glycosidase Assay::
100 units of β1-3 Galactosidase were incubated with 0.1 mM of flourescently-labeled oligosaccharides and glycopeptides, in a 10μl reaction for 20 hours at 37°C. The reaction products were analyzed by TLC for digestion of substrate.
No other glycosidase activities were detected (ND) with the following substrates:
β-N-Acetyl-glucosaminidase:
GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND
α-Fucosidase:
Fucα1-2Galβ1-4Glc-AMCGalβ1-4
(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND
α-Galactosidase:
Galα1-3Galβ1-4Gal-AMC ND
α-Neuraminidase:
Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ
1-4Glc-AMC ND
α-Mannosidase:
Manα1-3Manβ1-4GlcNAc-AMC
Manα1-6Manα1-6(Manα1-3)Man-AMC ND
β-Glucosidase:
Glcβ1-4Glcβ1-4Glc-AMC ND
β-Xylosidase:
Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND
β-Mannosidase:
Manβ1-4Manβ1-4Man-AMC ND
Endo F1, F2, H:
Dansylated invertase high mannose. ND
Endo F2, F3:
Dansylated fibrinogen biantennary. ND
PNGase F:
Fluoresceinated fetuin triantennary. ND
Protease Assay: :
After incubation of 100 units of β1-3 Galactosidase with 0.2 nmol of a standard mixture of proteins in a 20 µl reaction, for 20 hours at 37°C, no proteolytic activity could be detected by SDS-PAGE.
References
- Wong-Madden, S.T. and Landry, D. (1995) Glycobiology, 5, 19-28.
- Guthrie, E.P. and Taron, C.H., New England Biolabs, unpublished observations.
- Monks, B., New England Biolabs, unpublished observations.
- Taron, C.H., Benner, J.S., Horstra, L.J. and Guthrie, E.P. (1995) Glycobiology, 5, 603-610.
Reagents Sold Separately
BSA
Companion Products
Exoglycosidase Reaction Buffer Pack
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,770,405
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