New England Biolabs, P0745L, β1-4 Galactosidase S

β1-4 Galactosidase S is a highly specific exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing β1-4 linked galactose residues from oligosaccharides. 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, β-D-galactose from 1 nmol Galβ1-4GlcNAcβ1- 3Galβ1-4Glc-7-amino-4-methyl-coumarin (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: 231 kDa Specific Activity 123,000 units/mg Unit Assay Conditions Two fold dilutions of β1-4 Galactosidase 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 β1–4 Galactosidase and β1–4 Galactosidase S? A: β1–4 Galactosidase (NEB# P0730) is cloned from Bacteroides fragilis; whereas, β1–4 Galactosidase S (NEB# P0745) is cloned from Streptococcus pneumonia. Both enzymes cleave β1–4 linked galactose residues. The two versions of the enzyme have identical activity, specificity and concentration. Q: What is the difference between β1–4 Galactosidase S and β1–3 Galactosidase? A: β1–4 Galactosidase S will only cleave galactose residues with a β1–4 linked glycosidic bond, while β1–3 Galactosidase will cleave galactose residues with β1–3 and, at a lower efficiency, β1–6 linked glycosidic bonds. Q: What is a good positive control for β1–4 Galactosidase S? A: pNP-βGal (4-nitrophenyl-β-D-galactopyranoside) Q: Can I use β1–4 Galactosidase S in a double digest with other exoglycosidases and/or endoglycosidases? A: Yes. We recommend double digesting β1–4 Galactosidase 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 β1–4 Galactosidase S with PNGase F and/or O-Glycosidase. 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: Which enzyme should be used to remove β1-4 linked Galactose residues from intact IgG? A: β1-4 linked galactose residues can be difficult to remove from intact IgG under native conditions, and the activity varies greatly depending on the subtype of IgG. Extended incubations and increased units are usually needed to see substrate turnover. β1-4 Galactosidase S (NEB #P0745) is the preferred enzyme for this purpose as β1-4 Galactosidase (NEB #P0730) has very low activity on intact IgG. A protocol using up to 50*g of Rituximab (CHO derived IgG1) with 80 units (10μl) of β1-4 Galactosidase S (NEB #P0745 in 1X GlycoBuffer 1 in a total volume of 50μl, incubated for 24 hours at 37°C yields efficient removal of β1-4 galactose residues. However, similar conditions treating a murine IgG2a yields only ~70-80% removal of β1-4 galactose residues. 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.