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

FEBS Lett. 1994 Aug 1;349(2):173-80. doi: 10.1016/0014-5793(94)00647-4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Antibodies, Monoclonal / biosynthesis*
  • Antibody Specificity
  • Cattle
  • Epitopes / immunology
  • Fungal Proteins / chemistry
  • Fungal Proteins / immunology
  • Ligases / chemistry
  • Ligases / immunology
  • Rabbits
  • Saccharomyces cerevisiae Proteins*
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases
  • Ubiquitins / chemistry
  • Ubiquitins / immunology*

Substances

  • Antibodies, Monoclonal
  • Epitopes
  • Fungal Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitins
  • Adenosine Triphosphate
  • Ubiquitin-Conjugating Enzymes
  • UBR1 protein, S cerevisiae
  • Ubiquitin-Protein Ligases
  • Ligases