Quantitation of a guanine nucleotide binding regulatory protein by an enzyme-linked immunosorbent competition assay

We have developed a new method using a competitive enzyme-linked immunosorbent assay, ELISA, for the determination of levels of the stimulatory guanine nucleotide binding protein, Gs, in membrane extracts. The method is based on the use of antipeptide antibodies generated in rabbits directed against amino acids 28-42 in the alpha-subunit, alpha s, of Gs. The peptide is utilized as the stationary phase in the ELISA and anti-alpha s antibody bound to the microtiter plate is assessed by a peroxidase-coupled anti-rabbit immunoglobulin G antibody that yields detectable color development at 490 nm. Gs purified from rabbit liver is utilized as the standard to assess the ability of Gs present in cholate extracts of membrane samples to compete with bound peptides for primary antibody. This assay provides a direct means to quantify changes in levels of native Gs in membranes and cells.