Immunochemical and immunohistochemical localization of the G protein Gi1 in rat central nervous tissues

Antisera were raised in rabbits against purified alpha subunit of G protein Gi1 (Gi1 alpha) and also against a synthetic decapeptide corresponding to a sequence of Gi1 alpha. Antibodies in both antisera were purified with a Gi1-coupled Sepharose column, but purified anti-Gi1 alpha protein antibodies still reacted equally with both Gi1 alpha and Gi3 alpha, while anti-Gi1 alpha peptide antibodies reacted principally with Gi1 alpha. Using these antibodies, an enzyme immunoassay method for the quantification of Gi1 alpha was developed. The assay system consisted of polystyrene balls with immobilized anti-Gi1 alpha protein antibody F(ab')2 fragments and the anti-Gi1 alpha peptide antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 25 fmol of Gi1 alpha, and it did not cross-react with Gi2 alpha, Go alpha, or beta gamma. Samples from various regions of the rat central nervous system were homogenized in a 2% sodium cholate solution, and the concentration of Gi1 alpha in each extract was determined. Gi1 alpha was detected in all the regions, and the highest concentration was found in the olfactory bulb. Immunohistochemical study showed that Gi1 was mainly localized in the neuropil.