Source: Cloned from Streptococcus pneumoniae Molecular Weight: 74,000 daltons. α-Fucosidase: α2-3 and expressed in E. coli (1). Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND Quality Assurance: No contaminating Fucα1-2Galβ1-4Glc-AMC ND Neuraminidase S Supplied in: 50 mM NaCl, 20 mM Tris-HCl exoglycosidase or endoglycosidase F1, F2 or (pH 7.5 @ 25°C) and 1 mM EDTA. F3 activity could be detected. No contaminating β-Galactosidase: 1-800-632-7799 proteolytic activity could be detected. Galβ1-3GlcNAcβ1-4Galβ1-4Glc -AMC ND [email protected] Reagents Supplied with Enzyme: Galβ1-4GlcNAcβ1-3Galβ1-4Glc -AMC ND www.neb.com 10X GlycoBuffer 1 P0720S 015140716071 Quality Controls (0.5 M Sodium Acetate, pH 5.5 @ 25°C and Glycosidase Assays: 80 units of α2-3 α-Galactosidase: 50 mM CaCl ) Gal 1-3Gal 1-4Gal-AMC ND 2 Neuraminidase S were incubated with 0.1 mM α β

P0743S of flourescently-labeled oligosaccharides and Galα1-6Galα1-6Glcα1-2Fru-AMC ND Unit Definition: One unit is defined as the amount 400 units 8,000 U/ml Lot: 0011407 glycopeptides, in a 10 µl reaction for 20 hours at of enzyme required to cleave > 95% of the terminal 37°C. The reaction products were analyzed by TLC α-Mannosidase: RECOMBINANT Store at –20°C Exp: 7/16 α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1- for digestion of substrate. Manα1-3Manβ1-4GlcNAc-AMC Manα1-6Manα1-6(Manα1-3)Man-AMC ND Description: Neuraminidase is the common name 3GlcNAcβ1-3Galβ1-4Glc-AMC, in 1 hour at 37°C in a total reaction volume of 10 µl. for Acetyl-neuraminyl hydrolase (Sialidase). α2-3 No other glycosidase activities were detected (ND) α-Glucosidase: Neuraminidase S is a highly specific exoglycosi- with the following substrates: Unit Definition Assay:Two fold dilutions of 2-3 Glcα1-6Glcα1-4Glc-AMC ND dase that catalyzes the hydrolysis of α2-3 linked α N-acetyl-neuraminic acid residues from glycopro- Neuraminidase S are incubated with 1 nmol AMC- β-N-Acetylglucosaminidase: β-Xylosidase: teins and oligosaccharides. labeled substrate and 1X GlycoBuffer 1 in a 10 µl GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND reaction. The reaction mix is incubated at 37°C Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND Specificity: for 1 hour. Separation of reaction products are β-N-Acetylgalactosaminidase: β-Mannosidase: α(2–3) visualized via thin layer chromatography (2). GalNAcβ1-4Galβ1-4Glc-AMC ND R Manβ1-4Manβ1-4Man-AMC ND α(1–3) β(1–4) β(1–4) Specific Activity: ~160,000 units/mg. α-N-Acetylgalactosaminidase: (see other side) GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC ND NeuAc α(2–6)R = any sugarβ(1–4) β(1–2) α(1–3)

β(1–4) β(1–4) β(1–N) Asn CERTIFICATE OF ANALYSIS α(2–6) β(1–4) β(1–2) α(1–6) or α(1–6) α(1–3) α(1–3) β(1–4) β(1–6)

1-800-632-7799 [email protected] www.neb.com

CERTIFICATE OF ANALYSIS Endo F1, F2, H: Reaction Conditions: Optimal incubation times Notes on Use: References: Dansylated invertase high mannose. ND and enzyme concentrations must be determined • Reactions may be scaled-up linearly to 1. Chen, M. New England Biolabs, Inc., unpub- empirically for a particular substrate. Typical accommodate larger reaction volumes. lished results. Endo F , F : reaction conditions are as follows: 2 3 • The amount of exoglycosidase enzyme required 2. Wong-Madden, S. T. and Landry, D. (1995) Dansylated fibrinogen biantennary. ND 1. Combine 1 µg of glycoprotein or 100 nM of varies when different substrates are used. Start Glycobiology 5, 19–28. oligosaccharide and H 0 (if necessary) to make with 1–2 μl for 1 µg of glycoprotein or 100 nM Protease Assay: After incubation of 400 units of 2 a 9 µl total reaction volume. of oligosaccharide for one hour in a 10–25 μl α2-3 Neuraminidase S with 0.2 nmol of a standard reaction. If there is still undigested material, let mixture of proteins in a 20 µl reaction, for 20 hours 2. Add 1 μl of 10X GlycoBuffer 1 to make a 10 μl the reaction go overnight. ISO 9001 ISO 14001 ISO 13485 at 37°C, no proteolytic activity could be detected by total reaction volume. Registered Registered Registered Quality Environmental Medical Devices SDS-PAGE. Management Management 3. Add 1 µl of α 2-3 Neuraminidase S. NEW ENGLAND BIOLABS® is a registered trademark of New England Physical Purity: Purified to > 95% homogeneity as 4. Incubate at 37°C for 1 hour. Biolabs, Inc. determined by SDS-PAGE analysis using Coomassie Blue detection.

Heat Inactivation: 75°C for 10 minutes.

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