Monoclonal antibodies that distinguish between free and complexed heterotrimeric G protein beta subunits.
Heterotrimeric G proteins were purified from bovine brain by immunoaffinity chromatography on immobilized anti G protein monoclonal antibody 3C2. Release of betagamma subunits was effectuated by exposure of immobilized trimeric G proteins to MgAlF4. The resultant betagamma subunits were pure and biologically active. Following immunization of mice with purified betagamma subunits we obtained monoclonal anti beta antibodies showing broad species cross-reactivity. Characterization of the epitope recognized by one such monoclonal antibody, ARC9, indicated involvement of the extreme COOH-terminus, as assessed by its reactivity on beta subunits lacking the COOH-terminal 15 residues, obtained by in vitro translation. Although we used native betagamma subunits as immunogen, all monoclonal antibodies obtained failed to recognize assembled betagamma subunits, and were specific for free beta subunits. This property is useful in characterizing the assembly of G proteins from their subunits in living cells.
Rehm
A
A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Ploegh
H L
HL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000276:immunology; D015151:Immunoblotting; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins