O-glycosidase leaves only unsubstituted Gal-Beta(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 Endo-O-Glycosidase. At minimum, a sialadase such as Sialadase Au (alpha-2-3,6,8,9), part number E-S001, is almost always required to remove sialic acids
Source Recombinant Streptococcus pneumoniae in E.Coli
60 µl aliquot of enzyme (75 mU) in 50 mM sodium phosphate, pH 7.5.
Included with 20 µL and 60 µL pack sizes:
5x Reaction Buffer 5.0 (250 mM sodium phosphate, pH 5.0)
Specific Activity >12 U/mg
Activity >1.25 U/ml
Molecular weight 180,000 daltons
pH range 5-7, optimum 5.0
1. Add up to 100 µg of glycoprotein to a tube.
2. Add de-ionized water to a total of 13 µl.
3. Add 4 µl of 5x Reaction Buffer 5.0.
4. Add 1 µl of Sialadase Au (alpha 2-,3,6,8,9), part number E-S001.
5. Add 2 µl of O-Glycosidase.
6. Incubate for 1 hour at 37˚C.
Specific Activity 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- 2-acetamido-2-deoxy-3-O-(Beta-D-galactopyranosyl)-alpha- D-galactopyranoside.
Storage Store enzyme at 4˚C.
Stability Stable at least 12 months when stored properly. Several days exposure to ambient temperature will not reduce activity. Active for at lease 5 days under reaction conditions.
Purity O-Glycosidase is tested for contaminating protease as follows; 10 μg of denatured BSA is incubated for 24 hours at 37°C with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.
Bhavanandan, V.P., J. Umemoto and E.A. Davidson. Characterization of an endo-alpha-N-acetylgalactosaminidase from Diplococcus pneumoniae. Biochem Biophys 70: 738-74 5 (1976 ).
Fan, J.Q., K. Yamamoto, H. Kumagai and T. Tochikura. Induction and efficient purification of endo-alpha-N-acetyl- D-galactosaminidase from Alcaligenes sp. Agric Biol Chem 54: 233-23 4 (1990 ).
Glasgow, L R., J.C. Paulson and R.L. Hill. Systematic purification of five glycosidases from Streptococcus pneumoniae. J Biol Chem 252: 8615-8 623 (1977).
Iwase, H. and K . Hotta. Release of O-linked glycoprotein glycans by endo-alpha-N-acetyl-D-galactosaminidase. Meth Mol Biol 14: 151-159 (1993).
Unemoto, J., V.P. Bhavanandan and E.A. Davidson. Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J Biol Chem 252: 8609-8 614 (1977).