Monoclonal antibodies that distinguish between free and complexed heterotrimeric G protein β subunits

FEBS Letters

Ploegh, Hidde L¹  Rehm, Armin¹ 

[1] Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, 40 Ames Street, E 17-322, Cambridge, MA 02139, USA

关键词: Heterotrimeric G protein β subunit;    Monoclonal antibody;    Immunoaffinity chromatography;    Cross-reactivity;    Epitope recognition;    G proteins;    guanine nucleotide binding proteins;    mAb;    monoclonal antibody;    ELISA;    enzyme-linked immunosorbent assay;    GST;    glutathione S-transferase;    PAGE;    polyacrylamide gel electrophoresis;    Ig;    immunoglobulin;    ARK;    adrenergic receptor kinase;    BTK;    Bruton tyrosine kinase;    SDS;    sodium dodecyl sulfate;    NP-40;    Nonidet P-40;   

DOI  :  10.1016/S0014-5793(96)01457-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Heterotrimeric G proteins were purified from bovine brain by immunoaffinity chromatography on immobilized anti G protein monoclonal antibody 3C2. Release of βγ subunits was effectuated by exposure of immobilized trimeric G proteins to MgAlF₄. The resultant βγ subunits were pure and biologically active. Following immunization of mice with purified βγ subunits we obtained monoclonal anti β 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 β subunits lacking the COOH-terminal 15 residues, obtained by in vitro translation. Although we used native βγ subunits as immunogen, all monoclonal antibodies obtained failed to recognize assembled βγ subunits, and were specific for free β subunits. This property is useful in characterizing the assembly of G proteins from their subunits in living cells.

【 授权许可】

Unknown