Purified human inter-alpha inhibitor (IaI; Lane 1), purified pre-alpha-inhibitor (PαI; Lane 2), purified IαI incubated with recombinant full-length human TSG-6 (Q allotype; TSG-6Q; Lanes 3&4) with (Lane 3) or without (Lane 4) additional digestion with chondroitinase ABC lyase (Ch'ase for 4 h at 37˚C) were separated by SDS-PAGE and probed with 366921 (1:10,000 dilution). Proteins were visualized using an anti-rabbit IgG secondary antibody conugated to HRP and a chemiluminescence detection system. Note that incubating TSG-6 and IαI leads to the formation of IαI heavy chain (HC) and TSG-6 covalent adduct (HC·TSG-6) and the reaction by product HC·bikunin. The latter is degraded by chondroitinase ABC lyase to release free bikunin. In addition, HC·bikunin breaks down spontaneously to release bikunin·CS. (Image courtesy of Anthony J Day, University of Manchester, UK).