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Highly Sensitive Immunoassay for the α Subunit of the GTP‐Binding Protein Go and Its Regional Distribution in Bovine Brain
Author(s) -
Asano Tomiko,
Semba Reiji,
Ogasawara Nobuaki,
Kato Kanefusa
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb05710.x
Subject(s) - gtp' , protein subunit , immunoassay , distribution (mathematics) , chemistry , gtp binding protein regulators , g protein , biology , biochemistry , enzyme , antibody , receptor , immunology , gene , mathematical analysis , mathematics
Antisera were raised in rabbits against the α sub‐unit of a GTP‐binding protein, Go. Because the antisera cross‐reacted weakly with the α subunit of inhibitory GTP‐binding protein of adenylate cyclase (Gi), they were purified with a Goα‐coupled Sepharose column. Purified antibodies reacted only with Goα and did not cross‐react with the Giα subunit or βγ subunits in an immunoblot assay. Using these purified antibodies , a highly sensitive enzyme immunoassay method for the quantification of bovine brain Goα was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab′) 2 fragments and the same antibody Fab’ fragments labeled with β‐D‐galactosidase from Escherichia coli . The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Goa, and it did not cross‐react with Giα or βγ. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Goα were determined. Goα was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neu‐ ropil was rich in Goα .