Engineering and structural characterization of a linear polyubiquitin-specific antibody

J Mol Biol. 2012 May 4;418(3-4):134-44. doi: 10.1016/j.jmb.2011.12.053. Epub 2011 Dec 29.


Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry*
  • Antibodies / metabolism
  • Fluorescent Antibody Technique
  • Immunoglobulin Fab Fragments / chemistry*
  • Immunoglobulin Fab Fragments / metabolism
  • Immunoprecipitation
  • Models, Molecular
  • Polyubiquitin / chemistry*
  • Polyubiquitin / metabolism
  • Protein Conformation
  • Protein Engineering
  • Tumor Necrosis Factor-alpha / chemistry
  • Tumor Necrosis Factor-alpha / metabolism
  • Ubiquitination


  • Antibodies
  • Immunoglobulin Fab Fragments
  • Tumor Necrosis Factor-alpha
  • Polyubiquitin