Production and characterization of monoclonal antibodies specific to multi‐ubiquitin chains of polyubiquitinated proteins

FEBS Letters

Sawada, Hitoshi¹  Fujimuro, Masahiro¹  Yokosawa, Hideyoshi¹ 

[1] Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan

关键词: Ubiquitin;    Proteasome;    Multicatalytic;    Protease;    ATP;    Monoclonal antibody;    Ub;    ubiquitin;    PBS;    phosphate-buffered saline;    PAGE;    polyacrylamide gel electrophoresis;    DTT;    dithiothreitol;    IgG;    immunoglobulin G;    ELISA;    enzyme-linked immuno-sorbent assay;   

DOI  :  10.1016/0014-5793(94)00647-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Polyubiquitinated proteins tagged with multi-ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP-dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP-dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi-ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi-ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross-reacted with mono-ubiquitin and methyl-ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi-ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.

【 授权许可】

Unknown