Description
Neuraminidase Cp cleaves all non-reducing terminal non-branched α(2-3) and α(2-6) sialic acid residues from complex carbohydrates and glycoproteins.ContentsNeuraminidase Cp in 20 mM Tris-HCl, 25 mM NaCl, pH 7.5Included its 20 µL and 60 µL pack sizes:5x Reaction Buffer 250 mM sodium phosphate, pH 6.0Molecular weight41,000 daltonspH optimum6.0, active over the range 4.5-7.50 mM sodium phosphate (pH 6.0) provides the optimal buffer for enzyme activity with sialyllactose, a standard substrate. If glycosidase treatment is performed at suboptimal pH because of glycoprotein solubility or activity requirements, expect some diminution in enzyme activity.Specific ActivityOne unit of Neuraminidase is defined as the amount of enzyme required to produce 1 µmole of methylumbelliferone in 1 minute at 37°C, pH 5.0 from MU-NANA (2’-(4-methyl-umbelliferyl)-alpha-D-N acetylneuraminic acid]. Specificityα(2-3,6) Neuraminidase Cp cleaves all non- reducing terminal non-branched &alpha(2-3)- and α(2-6) sialic acid residues from complex carbohydrates and glycoproteins. There is no detectable activity on α (2-8) or α(2-9) linkages or on branched α(2-3) or α(2-6) linkages. The relative cleavage rates for different linkages are: α(2-3) > α(2-6).α(2-3,6) Neuraminidase Cp will not cleave branched sialic acids (linked to an internal residue). Use α(2-3,6,8,9) Neuraminidase for α(2-8) or branched sialic acids. To cleave only non-reducing terminal α(2-3) unbranched sialic acid residues, use α(2-3) Neuraminidase.α(2-3,6) Neuraminidase Cp is isolated from a clone of Clostridium perfringens. The enzyme has been extensively characterized using oligosaccharide standards.Relative activity a-(2-3) > a-(2-6)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.PurityNeuraminidase Cp 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 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 6.0.4. Add 2 µl Neuraminidase Cp.5. Incubate at 37°C for 1 hour.Desialylation may be monitored by SDS-PAGE if the size differential between native and de-sialylated protein is sufficient for detection