New England Biolabs, P0743L, α2-3 Neuraminidase S

α2-3 Neuraminidase S is a highly specific exoglycosidase that catalyzes the hydrolysis of α2-3 linked Related Categories Exoglycosidases Applications Expression Systems,, Glycan Sequencing,, Protein Digestion, Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3Galβ1- 3GlcNAcβ1-3Galβ1-4Glc-AMC, in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 1 Incubate at 37°C 1X GlycoBuffer 1 5 mM CaCl2 50 mM sodium acetate (pH 5.5 @ 25°C) Storage Buffer 50 mM NaCl 20 mM Tris-HCl 1 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Apparent: 74 kDa Specific Activity 160,000 units/mg Unit Assay Conditions Two fold dilutions of α2-3 Neuraminidase S are incubated with 1 nmol AMClabeled substrate and 1X GlycoBuffer 1 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 (1). FAQ Q: What are Glycosidases and their uses? A: Glycosidases are used to get information about the carbohydrate groups attached to glycoproteins and glycopeptides. They come in two varieties, endoglycosidases that cleave entire carbohydrate groups from proteins and exoglycosidases that remove monosaccharides from the non-reduced ends of the carbohydrate. A reduced end is one generated by an endoglycosidase. Researchers frequently use an endoglycosidase followed by one or more exoglycosidases and then analyze the products using SDS-PAGE or various types of liquid chromatography. Q: What is the difference between the four Neuraminidase enzymes sold by NEB: α2-3,6,8 Neuraminidase, α2-3,6,8,9 Neuraminidase A, α2-3 Neuraminidase and α2-3 Neuraminidase S? A: α2-3,6,8 Neuraminidase (NEB# P0720) is cloned from Clostridium perfringens and cleaves α2-3, α2-6 and α2-8 linked sialic acid residues. α2-3,6,8,9 Neuraminidase A (NEB# P0722) is cloned from Arthrobacter ureafaciens and cleaves α2-3, α2-6, α2-8 and α2-9 linked sialic acid residues. It can also cleave branched sialic acid residues that are linked to an internal residue. α2-3 Neuraminidase (NEB# P0728) is cloned from Salmonella typhimurium LT2 and cleaves α2-3 linked sialic acid residues. α2-3 Neuraminidase S (NEB# P0743) is cloned from Streptococcus pneumoniae and cleaves α2-3 linked sialic acid residues. Q: Can I use α2-3 Neuraminidase S in a double digest with other exoglycosidases and/or endoglycosidases? A: Yes. We recommend double digesting α2-3 Neuraminidase S with other exoglycosidases using 1X GlycoBuffer 1 (50mM NaAcetate, pH 5.5, 5mM CaCl2). We recommend using 1X G7 Reaction Buffer (50 mM sodium phosphate, pH 7.5) for digestion of α2-3 Neuraminidase S with PNGase F and/or O-Glycosidase. Q: What is a good positive control for α2-3 Neuraminidase S? A: Fetuin (NEB# P6042) or pNP-N-acetyl-neuraminic acid. Fetuin has both α2-3 and α2-6 terminal sialic acid residues; α2-3 Neuraminidase S will only cleave α2-3 linked residues, therefore the molecular weight change (due to sialic acid loss) will be intermediate if compared to treatment with a general neuraminidase. Q: Does this enzyme require denaturing conditions to act on glycoproteins? A: No, in general exoglycosidases can remove sugar residues from native (folded) glycoproteins (hydrophilic glycans face away from the protein backbone). However, the protein folding around a glycan site might affect enzyme accessibility. Exoglycosidase digestion of some proteins will require longer incubation times, or in some cases a mild extent of denaturation. Q: What is a good method to re-purify a glycan or glycopeptide after exoglycosidase treatment? A: Filtration with micro-spin filters, or solid-phase extraction (for example: graphitized carbon cartridges). Q: Do detergents inhibit glycosidases? A: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. Exceptions include PNGase F, PNGase F Recombinant, Remove-iT PNGase F and O-Glycosidase which are inhibited by SDS. Q: Do the NEB Neuraminidase enzymes cleave both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) residues? A: Depending on the enzyme source and specificity, some neuraminidases cleave both Neu5Ac and Neu5Gc, while some cleave only Neu5Ac. Those that cleave both Neu5Ac and Neu5Gc, tend to have lower efficiency towards Neu5Gc. NEB# P0720 (a2-3,6,8 Neuraminidase) – cleaves both Neu5Ac and Neu5Gc NEB# P0722 (a2-3,6,8,9 Neuraminidase A) – cleaves both Neu5Ac and Neu5Gc NEB# P0743 (a2-3 Neuraminidase S) – cleaves only Neu5Ac Q: How much exoglycosidase should be used? A: The amount of enzyme required varies when different substrates are used. Start with 1-2 µl for 1µg 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.