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O-Glycosidase & α2-3,6,8 Neuraminidase Bundle

Catalog # Concentration Size List Price Quantity Your Price
E0540S 0 $320.00
$288.00
Catalog # Size List Price Your Price
E0540S 0 $320.00
$288.00
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

This bundle contains 1 vial each of O-Glycosidase (NEB #P0733S) and a2-3,6,8 Neuraminidase (NEB #P0720S), which can be used simultaneously for the removal of terminal sialic acid residues and core 1 and core 3 O-glycans.

  • Recombinant enzymes with no detectable endoglycosidase or other exoglycosidase contaminating activities
  • Glycerol-free for optimal performance in HPLC and mass spectrometry analysis
  • ≥95% purity, as determined by SDS-PAGE and intact ESI-MS
  • Optimal activity and stability for up to 24 months

1 set of this bundle includes 2,000,000 units of O-Glycosidase and 2,000 units of Neuraminidase.

O-Glycosidase, also known as Endo-α-N-Acetylgalactosaminidase, catalyzes the removal of Core 1 and Core 3 O-linked disaccharides from glycoproteins. 

Neuraminidase is the common name for Acetyl-neuraminyl hydrolase (Sialidase). This Neuraminidase catalyzes the hydrolysis of α2-3, α2-6, and α2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and oligosaccharides. 




Product Source
O-Glycosidase is cloned from Enterococcus faecalis and expressed in E.coli (1). Neuraminidase is cloned from Clostridium perfringens (2) and overexpressed in E. coli (3).
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  α2-3,6,8 Neuraminidase P0720SVIAL -20 1 x 0.04 ml 50,000 units/ml
  O-Glycosidase P0733SVIAL -20 1 x 0.05 ml 4.0E7 units/ml
  GlycoBuffer 2 B3704SVIAL -20 1 x 1 ml 10 X
  NP-40 B2704SVIAL -20 1 x 1 ml 10 %
  Glycoprotein Denaturing Buffer B1704SVIAL -20 1 x 1 ml 10 X

Properties & Usage

Unit Definition

One unit of O-Glycosidase is defined as the amount of enzyme required to remove 0.68 nmol of O-linked disaccharide from 5 mg of Neuraminidase digested, non-denatured fetuin in 1 hour at 37°C in a total reaction volume of 100 µl (1 unit of both O-Glycosidase and PNGase F will remove equivalent molar amounts of O-linked disaccharides and N-linked oligosaccharides, respectively).

Non-denaturing Unit Definition of O-Glycosidase:
Two fold serial dilutions of O-Glycosidase are added to a reaction mixture of 5 mg of Neuraminidase digested fetuin with 1X GlycoBuffer 2. The reaction is then incubated at 37°C for 1 hour. O-linked disaccharide carbohydrates are determined by Morgan and Elson Assay (4). Note: Under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent.

Unit Definition of Neuraminidase:
One unit of Neuraminidase is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 5 minutes at 37°C in a total reaction volume of 10 µl.

1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT

1X NP-40
1% NP-40 in MilliQ-H2O

Reaction Conditions

1X GlycoBuffer 2
Incubate at 37°C

1X GlycoBuffer 2
50 mM Sodium Phosphate
(pH 7.5 @ 25°C)



Companion Products
Notes
  • Since O-Glycosidase is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time. Double digest with Endo H must have NP-40 present (NP-40 does not inhibit Endo H).
  • To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
References
  • Koutsioulis, D., Landry, D. and Guthrie, E.P. (2008). Glycobiology. 18, 799-805.
  • Roggentin, P. et al. (1988). FEBS Lett. 238, 31-34.
  • Guan, C. unpublished observations. New England Biolabs.
  • Morgan, W.T.J. and Elson, L.A. (1934). Biochem. J. 28, 988-995.
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
Legal And Disclaimer

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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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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