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α1-2,3,6 Mannosidase
Catalog #
P0768S
P0768L
Size
80 units
400 units
Concentration
2,000 units/ml
2,000 units/ml
Categories: Exoglycosidases, Proteome Analysis
Applications: Glycoprotein Production in Various Expression Systems, Removal of N-Linked & O-Linked Glycans from Glycoproteins,
Sequencing Glycans
Product
Information
FAQs &
Tech Tips
Protocols &
Manuals
Quality &
Safety
Description
Related Products
References
Legal
Information
Properties and Usage
Notes
Description
α1-2,3,6 Mannosidase, cloned from Jack Bean, and also known as JBM, is a broad specificity exoglycosidase that catalyzes the
hydrolysis of terminal α1-2, α1-3 and α1-6 linked mannose residues from oligosaccharides. α1-2,3,6 Mannosidase has a slight preference
for α1-2 mannose residues over α1-3 and α1-6 mannose residues.
Substrate Specificity
Product Source
Cloned from Canavalia ensiformis (Jack Bean) and expressed in Pichia pastoris (1).
Reagents Supplied
The following reagents are supplied with this product:
Store at (°C)
Concentration
GlycoBuffer 4
-20
10X
Zinc
-20
10X
Properties and Usage
Unit Definition
One unit is defined as the amount of enzyme required to cleave > 95% of the terminal mannose from 1 nmol of Man(α1,3)-Man(β1,4)-
GlcNAc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37°C in a total reaction volume of 10 µl.
Unit Assay Conditions:
Two fold dilutions of α1-2,3,6 Mannosidase are incubated with 1 nmol AMC-labeled substrate, 1X GlycoBuffer 4 and 1X Zinc in a 10 µl
reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via thin layer chromatography (2).
Reaction Conditions
1X GlycoBuffer 4
Supplement with 1X Zinc
Incubate at 37°C
1X GlycoBuffer 4:
50 mM sodium acetate
pH 4.5 @ 25°C
Storage Temperature
4°C
Storage Conditions
20 mM Tris-HCl
50 mM NaCl
pH 7.5 @ 25°C
Heat Inactivation
95°C for 10 min
Molecular Weight
Theoretical: 110 kDa
Related Products
Companion Products
α1-6 Mannosidase
α1-2,3 Mannosidase
Notes
1. α1-2,3,6 Mannosidase is a glycosylated protein. The theoretical molecular weight is 110 kDa, with two subunits at 66 kDa and 44 kDa
(3).
2. The optimal pH for α1-2,3,6 Mannosidase is 4.5. Enzyme activity is enhanced by the presence of 2 mM Zn2+ in the reaction.
3. In order to achieve complete removal of all non-reducing terminal α-linked mannose residues, it may be necessary to use an increased
enzyme concentration as well as an overnight incubation time (18 hours).
4. To achieve complete removal of α1-6 mannose residues from a complex substrate, a sequential digest using α1-2,3,6 Mannosidase
followed by α1-6 Mannosidase (NEB #P0727) may be necessary.
5. Reactions may be scaled-up linearly to accommodate larger reaction volumes.
6. The amount of exoglycosidase enzyme required varies when different substrates are used. Start with 1-2 μl for 1 μg of glycoprotein or
100 nM of oligosaccharide for one hour in a 10-25 μl reaction. If there is still undigested material, let the reaction go overnight.
References
1. Ganatra, M. New England Biolabs, Inc.. Unpublished observation
2. Wong-Madden, S.T. and Landry, D. (1995). Glycobiology. 5, 19-28.
3. Kumar, S.N. et al (2014). Glycobiology. 24, 252-261.
FAQs
Tech Tips
FAQs
1. What is the difference between α1-2,3 Mannosidase, α1-6 Mannosidase and α1-2,3,6 Mannosidase?
2. Can α1-2,3,6 Mannosidase (JBM) be a substrate itself for mannosidase activity?
3. When digesting complex substrates that include α1-6 Mannose residue, can additional mannose enzymes be used in
combination with α1-2,3,6 Mannosidase?
4. Does this enzyme require denaturing conditions to act on glycoproteins?
5. What is a good method to re-purify a glycan or glycopeptide after exoglycosidase treatment?
6. How much exoglycosidase should be used?
7. Do detergents inhibit glycosidases?
8. What are Glycosidases and their uses?
Tech Tips
Repeated freeze/thaw cycles may reduce activity. Recommended storage temperature is 4°C.
The optimal pH for α1-2,3,6 Mannosidase is 4.5. This differs from the majority of exoglycosidases, which have an optimal pH at
5.5.
Enzyme activity is enhanced by the presence of 2 mM Zn2+ in the reaction.
Using 1-2 µl is a good starting point for a 1 hr incubation of 1 µg of glycoprotein or 100 nM of oligosaccharide.
Protocols
Protocols
1. Typical Reaction Conditions for α1-2,3,6 Mannosidase (P0768)
Quality Control
Safety Data Sheet
Specifications
Quality Control
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.
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]
P0768S_L_v1
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
α1-2,3,6 Mannosidase
GlycoBuffer 4
Zinc
Legal and Disclaimers
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc
(NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes
in humans or animals.