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Exoglycosidases >
β-N-Acetylglucosaminidase |
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Substrate Specificity:
Description:
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. All reactions contained 4 units of β-N-Acetylglucosaminidase, 1X G1 Reaction Buffer 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.
Source:
Cloned from Xanthomonas manihotis and expressed in E. coli (1)
Reagents Supplied:
G1 Reaction Buffer (10X)
BSA (100X)
Enzyme Properties
Heat Inactivation:
65°C for 10 minutes
Molecular Weight:
Apparent: 71,000 daltons
Specific Activity:
50,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X G1 Reaction Buffer
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X G1 Reaction Buffer:
50 mM sodium citrate
pH 6.0 @ 25°C
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 G1 Reaction 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 (2).
Concentration:
4,000 units/ml
Storage Temperature:
4°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- Recommended storage temperature is 4°C.
- Avoid repeated freeze/thaw cycles.
FAQs
- Can I use β-N-Acetylglucosaminidase in a double digest with other exoglycosidases and/or endoglycosidases?
- What is the difference between β-N-Acetylglucosaminidase and β-N-Acetylhexosaminidasef?
- How much exoglycosidase should be used?
- Do detergents inhibit β-N-Acetylglucosaminidase?
- What is a good positive control for β-N-Acetylglucosaminidase?
- What are Glycosidases and their uses?
Protocols
Protocols for β-N-Acetylglucosaminidase
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 Assays:
16 units of β-N-Acetylglucosaminidase were incubated with 0.1 mM of flourescently-labeled oligosaccharides and glycopeptides, in a 10 µl 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-Acetylgalactosaminidase:
GalNacβ1-4Galβ1-4Glc-AMC ND
α-N-Acetylgalactosaminidase:
GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC ND
α-Fucosidase:
Fucα1-2Galβ1-4Glc-AMC ND
Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND
β-Galactosidase:
Galβ1-3GlcNAcβ1-4Galβ1-4Glc-AMC ND
Galβ1-4GlcNAcβ1-3Galβ1-4Glc-AMC ND
α-Galactosidase:
Galα1-3Galβ1-4Gal-AMC ND
Galα1-6Galα1-6Glcα1-2Fru-AMC ND
α-Neuraminidase:
Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-AMC ND
α-Mannosidase:
Manα1-3Manβ1-4GlcNAc-AMC ND
Manα1-6Manα1-6(Manα1-3)Man-AMC ND
β-Glucosidase:
Glcβ1-4Glcβ1-4Glc-AMC ND
α-Glucosidase:
Glcα1-6Glcα1-4Glc-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 28 units of β-N-Acetylglucosaminidase 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.
Physical Purity:
Purified to >95% homogeneity as determined by SDS-PAGE analysis using Coomassie Blue detection.
References
- Guthrie, E.P., Shimer, E.P., New England Biolabs, Inc., unpublished observations.
- Wong-Madden, S.T. and Landry, D. (1995) Glycobiology , 5, 19-28.
Reagents Sold Separately
BSA
Legal
Patents:
New England Biolabs, Inc.: 5,770,405
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England Biolabs, Inc. is an ISO 9001 and ISO
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