Product Description Recommended Reagents included with: 1 vial: 5x Reaction buffer 250 mM sodium phosphate, pH 5.0 Specific Activity One unit of O-Glycosidase 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-nitrophenyl2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)- alpha-D-galactopyranoside. Formulation The enzyme is provided as a sterile-filtered solution in 50 mM sodium phosphate (pH 7.5). Molecular Weight ~180,000 daltons pH Optimum 5, active over the range 5-7. Specifictity Cleaves only unsubstituted Gal-ß(1-3)GalNAc-alpha disaccharides attached to the serine or threonine residues of glycoproteins or glycopeptides. Substitutions such as sialic acid, galactose, fucose or N-acetylglucosamine must first be removed with the appropriate exoglycosidase prior to treatment with O-Glycosidase. At minimum, a neuraminidase such as Neuraminidase Au (Alpha-2-3,6,8,9), part number E-S001, is almost always required to remove sialic acids. Purity O-Glycosidase 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 use 1. Add up to 100 µg of glycoprotein to tube. 2. Add de-ionized water to a total of 13 µl. 3. Add 4 µl 5x Reaction Buffer 5.0. 4. Add 1 µl Neuraminidase AU (E-S001) 5. Add 2 µl O-Glycosidase. 6. Incubate at 37°C for 1 hour. Cleavage may be monitored by SDS-PAGE if the size differential between native and de-O-glycosylated protein is sufficient for detection.