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

Haopeng Wang, Atsushi Matsuzawa, Scott A. Brown, Jingran Zhou, Cliff S. Guy, Ping Hui Tseng, Karen Forbes, Thomas P. Nicholson, Paul W. Sheppard, Hans Häcker, Michael Karin, Dario A.A. Vignali

Research output: Contribution to journalArticlepeer-review

53 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)20197-20202
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number51
DOIs
Publication statusPublished - 2008 Dec 23
Externally publishedYes

Keywords

  • MAP kinase kinase kinase 1 (MEKK1)
  • TNF receptor-associated factor 6 (TRAF6)

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin'. Together they form a unique fingerprint.

Cite this