New England Biolabs, P0769S, α1-3,4 Fucosidase

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 α-fucose from 1 nmol of Galβ1-4GlcNAcβ1-3(Fucα1-3)Galβ1- 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 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 for 10 minutes Molecular Weight Apparent: 56 kDa Unit Assay Conditions Two fold dilutions of a1-3,4 Fucosidase are incubated with 1 nmol AMC-labeled substrate in 1X GlycoBuffer 1 supplemented with 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 is the difference between α1-2 Fucosidase, α1-3,4,6 Fucosidase and α1-3,4 Fucosidase? A: α1-2 Fucosidase (NEB# P0724) is cloned from Xanthomonas manihotis and is specific for the cleavage of linear α1-2 fucose residues. α1-2,4,6 Fucosidase (NEB# P0748) is cloned from bovine kidney and expressed in E. coli and is a broad specificity enzyme that catalyzes the hydrolysis of α1-2, α1-4, and α1-6 linked fucose residues. α1-3,4 Fucosidase (NEB# P0769) is cloned from the sweet almond tree (Prunus dulcis) and expressed in Pichia pastoris. α1-3,4 Fucosidase is a broad specificity enzyme that catalyzes the hydrolysis of α1-3 and α1-4 linked fucose residues from oligosaccharides and glycoproteins. Q: Can I use α1-3,4 Fucosidase in a double digest with other exoglycosidases and/or endoglycosidases? A: Yes. We recommend double digesting α1-3,4 Fucosidase with other exoglycosidases using 1X GlycoBuffer 1 (50mM NaAcetate, pH 5.5, 5mM CaCl2). To achieve optimal fucosidase 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 Fucosidase with PNGase F and/or O-Glycosidase. Q: Can α1-3,4 Fucosidase cleave core α1-3 fucose residues? A: No, α1-3,4 Fucosidase is unable to remove the core α1-3 fucose residue. However, it can cleave the α1-3 fucose residues located on the outer arm of N-linked glycans. Q: Can α1-3,4 Fucosidase and α1-2,3,4,6 Fucosidase cleave branched and complex substrates? A: α1-3,4 Fucosidase and α1-2,3,4,6 Fucosidase can cleave simple branched substrates to completion in 1 hour. However, a higher enzyme concentration and a longer incubation time (4 hours to overnight) may be needed for complete digestion of complex substrates with multiple branches. 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: 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: 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: 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: 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.