General Notes
Mouse anti pan ubiquitin antibody, clone P4D1 recognizes both mono- and polyubiquitin chains. The antibody has also been reported to recognize free polyubiquitin chains and free ubiquitin.Ubiquitin, as the name implies, is a ubiquitously expressed and highly conserved protein of 8.6 kDa. The protein is covalently linked to selected lysine residues in a post-translational modification process known as ubiquitylation or ubiquitination. This chemical reaction is mediated by three different protein families; ubiquitin-activating enzymes (also known as E1s), ubiquitin-conjugating enzymes (also known as E2s) and ubiquitin ligases (also known as E3s) (Hershko and Ciechanover 1998).The impact of ubiquitination depends on whether a single ubiquitin moiety (monoubiquitination) or an ubiquitin chain (polyubiquitination) has been attached to a protein. Monoubiquitination tends to trigger cellular processes related to endocytosis and membrane trafficking (Haglund et al. 2003) while the impact of polyubiquitination varies depending on how the ubiquitin residues in the chain have been linked. Attachment of Lysine-48 ubiquitin chains results in degradation by the 20S proteasome while addition of Lysine-63 ubiquitin chains mediates DNA damage and NFkappaB signaling (Chen 2005 and Mocciaro and Rape 2012). Lysine-6, Lysine-11, Lysine-27, Lysine-29 and Lysine-33 chains have also been reported (Komander 2009 and Ye and Rape 2011).When comparing staining of mouse anti pan ubiquitin antibody (clone P4D1) against staining with mouse anti polyubiquitin antibody (clone FK1) one can determine, if a protein target is mono- or polyubiquitinated. In contrast to mouse anti mono- and polyubiquitin antibody (FK2), clone P4D1 also recognizes free ubiquitin.