Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.
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.
Wang
Haopeng
H
Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.
Matsuzawa
Atsushi
A
Brown
Scott A
SA
Zhou
JingRan
J
Guy
Cliff S
CS
Tseng
Ping-Hui
PH
Forbes
Karen
K
Nicholson
Thomas P
TP
Sheppard
Paul W
PW
Häcker
Hans
H
Karin
Michael
M
Vignali
Dario A A
DA
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D019411:Clinical Laboratory Techniques; D008239:Lysine; D025821:Polyubiquitin / Q000032:analysis* / Q000276:immunology* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D054875:Ubiquitination
D000911:Antibodies, Monoclonal; D025821:Polyubiquitin; D008239:Lysine