Detailed Specificity: Source: Cloned from Arthrobacter ureafaciens and Molecular Weight: 100,000 daltons. α2-3,6,8,9 α2-3,6,8,9 Neuraminidase A will cleave branched expressed in E. coli (2). sialic acid residues that are linked to an internal resi- Quality Assurance: No contaminating Neuraminidase A due. This oligosaccharide from fetuin is an example Supplied in: 50 mM NaCl, 20 mM Tris-HCl (pH 7.5 @ exoglycosidase or endoglycosidase F1, F2 or of a side-branch sialic acid residue that can efficiently 25°C) and 1 mM EDTA. F3 activity could be detected. No contaminating 1-800-632-7799 be cleaved (1). proteolytic activity could be detected. [email protected] Reagents Supplied with Enzyme: www.neb.com 10X GlycoBuffer 1 α(2–3,6) β(1–4) β(1–2) Quality Controls P0722S 001150217021 (0.5 M Sodium Acetate, pH 5.5 @ 25°C and α Glycosidase Assays: 100 units of α2-3,6,8,9 (1–6) 50 mM CaCl ) 2 Neuraminidase A were incubated with 0.1 mM P0722S 2–3,6) β(1–4) β(1–4) of flourescently-labeled oligosaccharides and α( Unit Definition: One unit is defined as the amount 800 units 20,000 U/ml Lot: 0011502 α(2–3,6) β(1–3) glycopeptides, in a 10 µl reaction for 20 hours at β(1–4) ) of enzyme required to cleave > 95% of the terminal 37°C. The reaction products were analyzed by TLC (1–3 RECOMBINANT Store at –20°C Exp: 2/17 α α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1- for digestion of substrate. 3GlcNAc 1-3Gal 1-4Glc-AMC, in 1 hour at 37°C in Description: β β Neuraminidase is the common name α(2–3,6) β(1–4) No other glycosidase activities were detected (ND) (1–2) a total reaction volume of 10 µl. for Acetyl-neuraminyl hydrolase (Sialidase). β with the following substrates: α2-3,6,8,9 Neuraminidase A catalyzes the hy- β(1–4) β(1–2) α(1–6) Unit Definition Assay:Two fold dilutions of α2- drolysis of all linear and branched non-reducing α(1–3) β(1–4) β(1–4) -N-Acetylglucosaminidase: β(1–4) 3,6,8,9 Neuraminidase A are incubated with 1 nmol β terminal sialic acid residues from glycoproteins and GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND (2–6) (1–4) (1–2)AMC-labeled(1–3) substrate and 1X GlycoBuffer 1 in oligosaccharides. The enzyme releases α2-3 and β(1–4) Gal β(1–2)β(1–4)Man βα(1–4)(1–3)GlcNAcβ(1–6) β(1–6)NeuAc α R = any sugarβ β α a 10 µl reaction. The reaction mix is incubated at α2-6 linkages at a slightly higher rate than α2-8 β(1–4) β(1–4) β(1–4) β(1–4) β(1–N) Asn α(2–6) 37°C for 1 hour. Separation of reaction products are β-N-Acetylgalactosaminidase: and α2-9 linkages. β(1–4) β(1–2) α(1–6) β(1–3) β(1–3)β(1–3) β(1–3)or GalNAcβ1-4Galβ1-4Glc-AMC ND visualized via thin layer chromatographyα(1–6) (3). Specificity: α(2–3) α(1–3) α(2–6) α(1–3) β(1–4) β(1–6) Specific Activity: ~316,000 units/mg. >α(2–8) (see other side) >α(2–9) R CERTIFICATE OF ANALYSIS Detailed Specificity: Source: Cloned from Arthrobacter ureafaciens and Molecular Weight: 100,000 daltons. α2-3,6,8,9 α2-3,6,8,9 Neuraminidase A will cleave branched expressed in E. coli (2). sialic acid residues that are linked to an internal resi- Quality Assurance: No contaminating Neuraminidase A due. This oligosaccharide from fetuin is an example Supplied in: 50 mM NaCl, 20 mM Tris-HCl (pH 7.5 @ exoglycosidase or endoglycosidase F1, F2 or of a side-branch sialic acid residue that can efficiently 25°C) and 1 mM EDTA. F3 activity could be detected. No contaminating 1-800-632-7799 be cleaved (1). proteolytic activity could be detected. [email protected] Reagents Supplied with Enzyme: www.neb.com 10X GlycoBuffer 1 α(2–3,6) β(1–4) β(1–2) Quality Controls P0722S 001150217021 (0.5 M Sodium Acetate, pH 5.5 @ 25°C and α Glycosidase Assays: 100 units of α2-3,6,8,9 (1–6) 50 mM CaCl ) 2 Neuraminidase A were incubated with 0.1 mM P0722S 2–3,6) β(1–4) β(1–4) of flourescently-labeled oligosaccharides and α( Unit Definition: One unit is defined as the amount 800 units 20,000 U/ml Lot: 0011502 α(2–3,6) β(1–3) glycopeptides, in a 10 µl reaction for 20 hours at β(1–4) ) of enzyme required to cleave > 95% of the terminal 37°C. The reaction products were analyzed by TLC (1–3 RECOMBINANT Store at –20°C Exp: 2/17 α α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1- for digestion of substrate. 3GlcNAc 1-3Gal 1-4Glc-AMC, in 1 hour at 37°C in Description: β β Neuraminidase is the common name α(2–3,6) β(1–4) No other glycosidase activities were detected (ND) (1–2) a total reaction volume of 10 µl. for Acetyl-neuraminyl hydrolase (Sialidase). β with the following substrates: α2-3,6,8,9 Neuraminidase A catalyzes the hy- β(1–4) β(1–2) α(1–6) Unit Definition Assay:Two fold dilutions of α2- drolysis of all linear and branched non-reducing α(1–3) β(1–4) β(1–4) -N-Acetylglucosaminidase: β(1–4) 3,6,8,9 Neuraminidase A are incubated with 1 nmol β terminal sialic acid residues from glycoproteins and GlcNAcβ1-4GlcNAcβ1-4GlcNAc-AMC ND (2–6) (1–4) (1–2)AMC-labeled(1–3) substrate and 1X GlycoBuffer 1 in oligosaccharides. The enzyme releases α2-3 and β(1–4) Gal β(1–2)β(1–4)Man βα(1–4)(1–3)GlcNAcβ(1–6) β(1–6)NeuAc α R = any sugarβ β α a 10 µl reaction. The reaction mix is incubated at α2-6 linkages at a slightly higher rate than α2-8 β(1–4) β(1–4) β(1–4) β(1–4) β(1–N) Asn α(2–6) 37°C for 1 hour. Separation of reaction products are β-N-Acetylgalactosaminidase: and α2-9 linkages. β(1–4) β(1–2) α(1–6) β(1–3) β(1–3)β(1–3) β(1–3)or visualized via thin layer chromatography (3). GalNAcβ1-4Galβ1-4Glc-AMC ND Specificity: α(1–6) α(2–3) α(1–3) α(2–6) α(1–3) β(1–4) β(1–6) Specific Activity: ~316,000 units/mg. >α(2–8) (see other side) >α(2–9) R CERTIFICATE OF ANALYSIS α-N-Acetylgalactosaminidase: β-Xylosidase: Reaction Conditions: Optimal incubation times References: GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC ND Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND and enzyme concentrations must be determined 1. Iwamori, M., Ohta, Y., Uchida, Y. and Tsukada, empirically for a particular substrate. Typical Y. (1997) Glycocon. J., 1, 67–73. α-Fucosidase: β-Mannosidase: reaction conditions are as follows: 2. McLeod, E. New England Biolabs, Inc., unpub- Galβ1-4 (Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-AMC ND Manβ1-4Manβ1-4Man-AMC ND 1. Combine 1 µg of glycoprotein or 100 nM of lished results. Fucα1-2Galβ1-4Glc-AMC ND oligosaccharide and H 0 (if necessary) to make Endo F , F , H: 2 3. Wong-Madden, S. T. and Landry, D. (1995) 1 2 a 9 µl total reaction volume. Glycobiology 5, 19–28. β-Galactosidase: Dansylated invertase high mannose. ND Galβ1-3GlcNAcβ1-4Galβ1-4Glc -AMC ND 2. Add 1 µl of 10X GlycoBuffer 1 to make a 10 µl total reaction volume. Galβ1-4GlcNAcβ1-3Galβ1-4Glc -AMC ND Endo F2, F3: Dansylated fibrinogen biantennary. ND 3. Add 1 µl of α2-3,6,8,9 Neuraminidase A. ISO 9001 ISO 14001 ISO 13485 α-Galactosidase: Registered Registered Registered Quality Environmental Medical Devices Galα1-3Galβ1-4Gal-AMC ND Protease Assay: After incubation of 1,000 units 4. Incubate at 37°C for 1 hour. Management Management Gal 1-6Gal 1-6Glc 1-2Fru-AMC ND of 2-3,6,8,9 Neuraminidase A with 0.2 nmol of a α α α α NEW ENGLAND BIOLABS® is a registered trademark of New England standard mixture of proteins in a 20 µl reaction, for Notes on Use: Biolabs, Inc. α-Mannosidase: 20 hours at 37°C, no proteolytic activity could be • Reactions may be scaled-up linearly to Manα1-3Manβ1-4GlcNAc-AMC ND detected by SDS-PAGE. accommodate larger reaction volumes. Manα1-6Manα1-6(Manα1-3)Man-AMC ND • The amount of exoglycosidase enzyme required Physical Purity: Purified to > 95% homogeneity as varies when different substrates are used. Start α-Glucosidase: determined by SDS-PAGE analysis using Coomassie with 1–2 µl for 1 µg of glycoprotein or 100 nM Glcα1-6Glcα1-4Glc-AMC ND Blue detection. of oligosaccharide for one hour in a 10–25 µl reaction. If there is still undigested material, let Heat Inactivation: 75°C for 10 minutes. the reaction go overnight. • Higher concentrations of enzyme as well as longer incubation times may be necessary for Page 2 (P0722) cleavage of branched structures. α-N-Acetylgalactosaminidase: β-Xylosidase: Reaction Conditions: Optimal incubation times References: GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC ND Xylβ1-4Xylβ1-4Xylβ1-4Xyl-AMC ND and enzyme concentrations must be determined 1.