Detection of E3-independent polyubiquitination of AtUBC22 by luminescent analysis. A, Polyubiquitin chain on AtUBC22 but not on AtUBC35 was detected by immunoblot analysis. In this assay, polyubiquitination reaction was carried out with FLAG-tagged ubiquitin, and detected by immunoblot analysis using anti-FLAG antibody. B, Schematic diagram of detection of polyubiquitin chains by luminescent analysis. Protein A-conjugated acceptor beads and streptavidin-coated donor beads are bound to anti-FLAG antibody bound to FLAG-tagged ubiquitin and biotinylated E2, respectively, and these two beads are in closed proximity when polyubiquitin chain formed. Upon excitation 680 nm, a singlet oxygen is generated from the donor beads, and then transferred to the acceptor beads within 200 nm, and the singlet oxygen reacts the acceptor beads which in turn emits light at 520–620 nm. This light is measured by AlphaScreen kit and change to signal value. C, Polyubiquitin chain on purified recombinant E2 was detected by luminescent analysis in the presence (E1 +) or absence (E1 -) of exogenous E1. Error bars represent standard deviations from three independent experiments.