Open AccessAnalysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin
Author(s) -
Haopeng Wang,
Atsushi Matsuzawa,
Scott A. Brown,
Jingran Zhou,
Cliff Guy,
PingHui Tseng,
Karen Forbes,
Thomas P. Nicholson,
Paul W. Sheppard,
Hans Häcker,
Michael Karin,
Dario A.A. Vignali
Publication year - 2008
Publication title -
proceedings of the national academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0810461105
Subject(s) - lysine , monoclonal antibody , endocytosis , microbiology and biotechnology , ubiquitin , signal transduction , ex vivo , in vitro , immunoprecipitation , biology , blot , in vivo , receptor , biochemistry , antibody , chemistry , immunology , amino acid , genetics , gene
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.
Accelerating Research
Robert Robinson Avenue,
Oxford Science Park, Oxford
OX4 4GP, United Kingdom
Address
John Eccles HouseRobert Robinson Avenue,
Oxford Science Park, Oxford
OX4 4GP, United Kingdom