Schematic representation of the developed assays A. TUBE-AlphaLISA assay. Ubiquitylated proteins from cell lysates are captured by the GST-TUBE trap during the lysis. The GST-TUBEs trap binds to GSH AlphaLISA acceptor beads. FK2 antibody recognizes and binds to ubiquitin chains covalently attached to substrates and it is recognized by the protein A AlphaLISA donor bead. AlphaLISA donor bead is excited at 680 nm releasing singlet oxygen that diffuses towards the acceptor bead. After excitation acceptor bead emits light at 615 nm. B. Schematic representation of TUBE-DELFIA assay
P. falciparum iRBCs are lysed in presence of biotin-TUBEs attached to streptavidin coated plates. Ubiquitylated proteins from cell lysates captured by the TUBEs ubiquitin traps are then immunodetected by the anti-ubiquitin FK2 mouse antibody which is recognized by a secondary antimouse antibody labeled with europium. The addition of the enhancement solution causes the release of the europium generating signal at 615 nm. Washes are required before each incubation step to remove unbound proteins/reagents.