Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20197-202. doi: 10.1073/pnas.0810461105. Epub 2008 Dec 17.


Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal*
  • Antibody Specificity
  • Clinical Laboratory Techniques
  • Lysine*
  • Polyubiquitin / analysis*
  • Polyubiquitin / immunology*
  • Polyubiquitin / isolation & purification
  • Protein Processing, Post-Translational
  • Ubiquitination*


  • Antibodies, Monoclonal
  • Polyubiquitin
  • Lysine