New England Biolabs, P0746S, β1-3,4 Galactosidase

β1-3,4 Galactosidase is an exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing β1-3 and β1-4-linked galactose residues from oligosaccharides. Related Categories Exoglycosidases Applications Glycan Sequencing,, Recombinant Glycoprotein Expression,, Glycoprotein Analysis 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 4 Incubate at 37°C 1X GlycoBuffer 4 50 mM sodium acetate (pH 4.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 1 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C for 10 minutes Molecular Weight Theoretical: 71 kDa Unit Assay Conditions Two fold dilutions of β1-3,4 Galactosidase are incubated with 1 nmol AMC-labeled substrate and 1X GlycoBuffer 4 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 is the difference between β1-4Galactosidase, β1-4Galactosidase S and β1-3,4Galactosidase? A: β1-4 Galactosidase (NEB# P0730 ) is cloned from Bacteroides fragilis; β1-4 Galactosidase S (NEB# P0745 ) is cloned from Streptococcus pneumonia, and β1-3,4 Galactosidase (NEB# P0746 ) is cloned from bovine testis. β1-4 Galactosidase and β1-4 Galactosidase S cleave only β1-4 linked galactose residues. Whereas, β1-3,4 Galactosidase (BTG) cleaves both β1-3 and β1-4 linked galactose residue. Q: Can β1-3,4Galactosidase cleave β1-6 linked galactose residues? A: Although rare in nature, β1-6 linked galactose residues have been shown to be hydrolyzed by β1-3,4 Galactosidase at a much slower rate than β1-3 or β1-4 linked galactose residues. Q: What is a good positive control for β1-3,4 Galactosidase? A: pNP-βGal (4-nitrophenyl-β-D-galactopyranoside) Q: Can I use β1-3,4 Galactosidase in a double digest with other exoglycosidases and/or endoglycosidases? A: Yes. Although the optimal buffer for β1-3,4 Galactosidase is 1X Glycobuffer 4 (50mM sodium acetate, pH 4.5), we recommend double digesting β1-3,4 Galactosidase with other exoglycosidases using 1X GlycoBuffer 1 (50mM NaAcetate, pH 5.5, 5mM CaCl2). We recommend using 1X Glycobuffer 2 (50 mM sodium phosphate, pH 7.5) for digestion of β1-3,4 Galactosidase with PNGase F and/or O-Glycosidase. 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: 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. 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: 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: 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.