New England Biolabs, P0747S, α1-3,4,6 Galactosidase

Related Categories Exoglycosidases,, Proteome Analysis Applications Expression Systems,, Glycan Sequencing,, Proteomics, Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of the terminal, α-D-galactose from 1 nmol Galα1-3Galβ1-4Gal- 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 Supplement with 100 µg/ml Recombinant Albumin (Low-glycerol) Incubate at 37°C 1X GlycoBuffer 1 5 mM CaCl2 50 mM sodium acetate (pH 5.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl 1 mM EDTA pH 7.5 @ 25°C Heat Inactivation 65°C Molecular Weight Theoretical: 39.7 kDa Unit Assay Conditions Two fold dilutions of α1-3,4,6 Galactosidase are incubated with 1 nmol AMC labeled substrate in 1X GlycoBuffer 1 and 1X rHSA 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 α1-3,6 Galactosidase and α1-3,4,6 Galactosidase? A: α1-3,6 Galactosidase (NEB# P0731) is cloned from Xanthomonas manihotis and is specific for the cleavage of only α1-3 and α1-6 galactose residues. α1-3,4,6 Galactosidase (NEB# P0747) is cloned from green coffee bean and is a broader specificity enzyme that catalyzes the hydrolysis of α1-3, α1-4, and α1-6 linked galactose residues. Q: Can I use α1-3,4,6 Galactosidase in a double digest with other exoglycosidases and/or endoglycosidases? A: Yes. We recommend double digesting α1-3,4,6 Galactosidase with other exoglycosidases using 1X GlycoBuffer 1 (50mM NaAcetate, pH 5.5, 5mM CaCl2). To achieve optimal galactosidase activity, supplement the reaction with rHSA. We recommend using 1X GlycoBuffer 2 (50 mM sodium phosphate, pH 7.5) for digestion of α1-3,4,6 Galactosidase with PNGase F and/or O-Glycosidase. Q: What is a good positive control for α1-3,4,6 Galactosidase? A: p-Nitrophenyl α-D-galactopyranoside (alternative name: 4-Nitrophenyl α-D-galactopyranoside) 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: 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.