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.