A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.
A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.
Kawashima
H
H
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
Murata
T
T
Yamamoto
K
K
Tateishi
A
A
Irimura
T
T
Osawa
T
T
D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry / Q000302:isolation & purification; D001216:Asparagine / Q000737:chemistry; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D001212:Asialoglycoproteins; D006023:Glycoproteins; D006834:Hydrazines; D007074:Immunoglobulin G; D009844:Oligosaccharides; D009961:Orosomucoid; C017030:asialoorosomucoid; D012967:Sodium Dodecyl Sulfate; D001216:Asparagine