N-Acetylglucosaminidase (C09-0992-478)

Product DescriptionN-acetylglucosaminidase cleaves all non-reducing terminal β-linked N-acetylglucosamine residues from complex carbohydrates and glycoproteins.The cleavage rates of different linkages of GlcNAc on bi-, tri- and tetraantennary oligosaccharides is greatly dependent on the steric hindrance by neighboring residues. The β(1-2)GlcNAc residue linked to the β(1-3)-linked mannose is cleaved at the highest rate and the β(1-2) GlcNAc residue linked to the β(1-6)-linked mannose at the lowest rate for all three oligosaccharides. The β(1-6) GlcNAc residue, when present, is removed at the second highest rate and the β(1-4) GlcNAc, third. On a triantennary structure, this residue is removed at the second highest rate. A bisecting β(1-4) GlcNAc linked to the β-linked mannose severely hinders cleavage of other GlcNAc residues–high concentrations of enzymes and prolonged incubation times are required for cleavage.ContentsN-Acetylglucosaminidase in 20 mM Tris-HCl, 25 mM NaCl (pH 7.5).included with 20 µL and 60 µL pack sizes:1 vial: 5x Reaction buffer250mM Sodium phosphate, pH 5Molecular Weight~140,000 daltonspH optimum5.0, active over the range 5-7SpecificityAll non-reducing terminal ß-linked N-acetylglucosamine. Bisecting GlcNAc slows the reaction.Specific Activity AssayOne unit of N-acetylglucosaminidase is defined as the amount of enzyme required to produce 1 µmole of p-nitrophenol (pNP) in 1 minute at 37°C, pH 5.0 from p-nitrophenyl-ß-D-N-acetyl-glucosaminide.FormulationThe enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl, 25 mM NaCl (pH 7.5).StabilityStable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity.PurityN-acetylglucosaminidase is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.Directions for use1. Add up to 100 µg of asialogalacto-glycoprotein or 1 nmol of oligosaccharide to tube.2. Add de-ionized water to a total of 14 µl.3. Add 4 µl 5x Reaction Buffer 5.0.4. Add 2 µl N-acetylglucosaminidase5. Incubate at 37°C for 3 hours. If bisecting GlcNAc is present, incubation time should be increased to 12 hours.Progress may be monitored by SDS-PAGE if the size differential between native and de-glycosylated protein is sufficient for detection.  Application•Structural analysis of oligosaccharides•Distinguishing different N-acetyl glucosamine link-ages•Distinguishing between N-acetyl glucosamine and N-acetylgalactosamine•Removing heterogeneity from glycoproteins. Specifications and Purity: Specific Activity>80 U/mg;Activity>50 U/ml. Related Document: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/N489924-SCI_2299096f9b4fbd5e4cca9a4652014cf0.pdf.