title: Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.
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.
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.
authors.forename: Haopeng
authors.initials: H
authors.lastname: Wang
authors.forename: Atsushi
authors.initials: A
authors.lastname: Matsuzawa
authors.forename: Scott A
authors.initials: SA
authors.lastname: Brown
authors.forename: JingRan
authors.initials: J
authors.lastname: Zhou
authors.forename: Cliff S
authors.initials: CS
authors.lastname: Guy
authors.forename: Ping-Hui
authors.initials: PH
authors.lastname: Tseng
authors.forename: Karen
authors.initials: K
authors.lastname: Forbes
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Nicholson
authors.forename: Paul W
authors.initials: PW
authors.lastname: Sheppard
authors.forename: Hans
authors.initials: H
authors.lastname: Häcker
authors.forename: Michael
authors.initials: M
authors.lastname: Karin
authors.forename: Dario A A
authors.initials: DA
authors.lastname: Vignali
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D025821:Polyubiquitin; D008239:Lysine