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

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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.

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