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Exoglycosidases >
α1-6 Mannosidase |
 | | | | α1-6 Mannosidase | | | Concentration and Specificity Changed | |
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Prices are in US dollars and valid only for US orders.
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Substrate Specificity:
Description:
α1-6 Mannosidase is a highly specific exoglycosidase that removes unbranched α1-6 linked D-mannopyranosyl residues from oligosaccharides (1,2). When used in conjunction with α1-2,3 Mannosidase, the α1-6 Mannosidase will cleave α1-6 Mannose residues from branched carbohydrate substrates.
Figure 1: Detailed specificity of α1-6 Mannosidase. All reactions contained 32 units of α1-2,3 Mannosidase (NEB #P0729), 40 units of α1-6 Mannosidase, 1X G6 reaction buffer and 1X BSA In a total reaction volume of 10 µl. Reactions were incubated at 37°C. The substrate depicted in (E) will not cut to completion. If this structure exists in any substrate it will be impervious to cleavage by α1-6 Mannosidase. Note: When used alone, α1-6 Mannosidase will still act only on linear substrates. When used in conjunction with α1-2,3 Mannosidase, the α1-6 Mannosidase will cleave α1-6 Mannose residues from branched carbohydrate substrates.
Source:
Cloned from Xanthomonas manihotis and expressed in E. coli (2).
Reagents Supplied:
G2 Reaction Buffer (10X)
BSA (100X)
Enzyme Properties
Molecular Weight:
Apparent: 51,000 daltons
Specific Activity:
136,000 units/mg
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 α-D-mannose from 1 mol of Manα1-6Manα1-6Man-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 α1‑6 Mannosidase are incubated with 1 nmol AMC-labeled substrate in 1X G2 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 (1).
Concentration:
40,000 units/ml
Storage Conditions:
20 mM Tris-HCl
50 mM NaCl
0.1 mM EDTA
0.006% sodium azide
100 µg/ml BSA
pH 7.5 @ 25°C
Storage Temperature:
4°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- p-nitrophenyl-α-D-mannopyranoside is NOT a substrate for this enzyme.
- A double digest with α1-2,3 Mannosidase requires the following reaction conditions: 1X G6 Reaction Buffer: 50 mM Sodium Acetate (pH 5.5 @ 25°C), 5 mM CaCl2. Supplement with 100 µg/ml BSA. Incubate at 37°C.
FAQs
- How much exoglycosidase should be used?
- Do detergents inhibit exoglycosidases/endoglycosidases?
- What is a good positive control for α1-6 Mannosidase?
- 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 (ND).
Glycosidase Assay:
80 units of α1‑6 Mannosidase 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-Acetylglucosaminidase:
GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND
a-N-Acetylgalactosaminidase:
GalNAca1-3(Fucα1-2)Galβ1-4Glc-AMC ND
α-Fucosidase:
Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND
Fucα1-2Galβ1-4Glc-AMC ND
α-Galactosidase:
Galα1-3Galβ1-4Gal-AMC ND
b-Galactosidase:
Galb1-3GlcNAcβ1-4Galβ1-4Glc-AMC ND
Galb1-4GlcNAcβ1-3Galβ1-4Glc-AMC ND
α-Neuraminidase:
Neu5Acα2-3Galβ1-3GlcNAcβ1-3 Galβ1-4Glc-AMC ND
α-Mannosidase:
Manα1-3Manβ1-4GlcNAc-AMC ND
β-Glucosidase:
Glcβ1-4Glcβ1-4Glc-AMC ND
a-Glucosidase:
Glca1-6Glca1-4Glc-AMC
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 560 units of α1‑6 Mannosidase with 0.2 nmol of a standard mixture of proteins 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.
Reagents Sold Separately
BSA
Companion Products
Exoglycosidase Reaction Buffer Pack
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 7,094,563
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England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
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