Fig. 2
From: Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay
Applications of the UPS-CONA assay. UPS-CONA assay can be used to monitor various ubiquitin-related reactions. For example, ubiquitin conjugation to E1 and E2 enzymes, HECT or RBR E3 ligases and substrate proteins can be followed by immobilizing the proteins or enzymes of interest on polymer microbeads and incubating them with appropriate reaction components and with a fluorescently labeled ubiquitin (red). Increase in bead fluorescence will indicate a successful reaction (a). Ubiquitin release from an on-bead substrate and transfer to a downstream cascade component can be detected as a decrease of bead fluorescence (b). Ubiquitin transfer between different components of a cascade can also be observed (c)