Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins.
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.
Fujimuro
M
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Sawada
H
H
Yokosawa
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D002417:Cattle; D000939:Epitopes / Q000276:immunology; D005656:Fungal Proteins / Q000737:chemistry / Q000276:immunology; D008025:Ligases / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D008025:Ligases