Ubiquitination assay using fluorescently labelled ⁸⁰⁰Ub (green) and ⁶⁸⁰mG2-pUb (red) to test the activity of (A) parkin and two truncated parkin species, R0RBR and ⁷⁷R0RBR. The left panel shows ubiquitination in the absence of mG2-pUb whereas the centre and right panels show ubiquitination experiments filtered for ubiquitination of mG2-pUb (red) and overall ubiquitination (green), respectively. Ubiquitination of mG2-pUb (red) and overall ubiquitination (green) using (B) parkin^S65A and (C) parkin^K211N. Reactions were initiated by addition of Uba1 (E1) and quenched at the required timepoints with 3× sample buffer and DTT.