A method for assessing the reassembly of a multisubunit glycoprotein by western blotting.
The Western blot procedure has been adapted to detect the reassembly of a two-subunit glycoprotein, urinary human chorionic gonadotropin (hCG), directly on the nitrocellulose. This glycoprotein is composed of two nonidentical subunits, alpha and beta. A simple procedure using immunoblotting has been developed to detect reassembly of the monomers to dimer. Three monoclonal antibodies were required for the development of this method: A109, which binds the alpha subunit or hCG; B105, which binds the beta subunit or hCG; and B107, specific for the intact hCG dimer. The alpha subunit and beta subunit of hCG were each electrophoresed and transferred to nitrocellulose, and the transfer was then incubated with the appropriate complementary subunit; reassembly of the dimer was determined by the binding of the monoclonal antibody B107. Evidence that the reassembly occurs directly on the nitrocellulose comes from the fact that B107 immunoreactivity is detected at the molecular weight position of the subunit and not at the dimer molecular weight. A genetically expressed recombinant form of the alpha subunit was also tested for its ability to recombine with the opposite subunit to produce the dimer. The recombinant alpha subunit was determined to have additional carbohydrate which interfered with the binding of the beta subunit. N-Glycanase digestion of the recombinant alpha subunit produced a form which, when incubated with the beta subunit, did recombine on the nitrocellulose and could be recognized by B107.
Lustbader
J W
JW
Department of Medicine, College of Physicians & Surgeons of Columbia University, New York, New York 10032.
Pollak
S
S
D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006026:Glycoside Hydrolases; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D006023:Glycoproteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C020668:glycanase