Faculty Collaboration Database

Characterization and use of crude alpha-subunit preparations for quantitative immunoblotting of G proteins. Anal Biochem 1994 Jul;220(1):82-91

Date

07/01/1994

Pubmed ID

7978261

DOI

10.1006/abio.1994.1302

Scopus ID

2-s2.0-0028178975 (requires institutional sign-in at Scopus site)   45 Citations

Abstract

G proteins are heterotrimeric membrane-associated proteins that couple a large number of receptors to a variety of effector systems within the cell. Characterization of G proteins expressed in a particular cell type represents an important first step in defining the potential candidates to which a receptor might couple. A difficulty often encountered using G protein antisera from various commercial and private sources is relating the intensity of bands on a Western blot to the relative amount of G protein present in a membrane preparation. This problem is especially noteworthy when comparing across G protein subtypes due to differences in titer, affinity, and specificity among various antisera. Conventional approaches to obtaining G protein standards of sufficient purity to address these issues in a quantitative manner are time-consuming and difficult, but the procedures outlined herein demonstrate a method for using DEAE fractions from Escherichia coli expressing individual alpha-subunits. The key features of the present approach are to estimate saturable GTP gamma S binding in each alpha-subunit preparation and calculate the moles of alpha-subunit present in the respective preparations based on the known stoichiometry of GTP gamma S binding (1:1). The extent of correspondence between GTP gamma S binding and immunoreactivity is then determined by trypsin protection assays, which estimate the proportion of immunodetectable G protein which can bind GTP gamma S. After characterization in this manner, DEAE fractions from bacteria transformed with the respective cDNA for Gi alpha-1, G1 alpha-2, and G1 alpha-3 were used to construct standard curves on Western blots and estimate endogenous G protein concentrations in cell lines (CHO and HeLa) and across species (rat and mouse) in isolated adipocyte preparations. Plasma membranes from CHO cells contained Gi alpha-2 (4.8 +/- 0.3 pmol/mg protein) and Gi alpha-3 (0.6 +/- 0.1 pmol/mg protein), but not Gi alpha-1, while HeLa cell membranes contained Gi alpha-1 (0.11 +/- 0.01 pmol/mg protein) and Gi alpha-3 (1.3 +/- 0.1 pmol/mg protein), but not Gi alpha-2. In contrast, rat and mouse adipocyte membranes contained Gi alpha-1 (48 +/- 2 vs 36 +/- 2 pmol/mg protein), Gi alpha-2 (77 +/- 1.5 vs 25 +/- 1.4 pmol/mg protein), and Gi alpha-3 (26 +/- 1.2 vs 15 +/- 1 pmol/mg protein). The method described herein provides an innovative solution to the technically difficult problem of obtaining pure standards for the assay of G protein alpha-subunits and does so using simple biochemical and immunological techniques.

Author List

Gettys TW, Sheriff-Carter K, Moomaw J, Taylor IL, Raymond JR

Author

John R. Raymond MD President, CEO, Professor in the President department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Antibody Specificity
CHO Cells
Cells, Cultured
Cricetinae
GTP-Binding Proteins
HeLa Cells
Humans
Immunoblotting
Male
Mice
Peptide Fragments
Rats
Reference Standards
Trypsin