Preparation of multisite ubiquitinated proteins

For multisite ubiquitinated α-synuclein with the same Ub chain linkages (here, monoUb), 1 μM Uba1, 20 μM UBE2E1, 8 μM Fluorescent-labeled α-synuclein containing two SUE1 tags, 40 μM monoUb were mixed and reacted in the reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl₂ and 10 mM ATP) at 37 °C for 2 h. For multisite ubiquitinated α-synuclein with different Ub chain linkages (here K48-linked diUb modified at K43 site, monoUb modified at K96 site), 1 μM Uba1, 40 μM UBE2E1, 16 μM Fluorescent-labeled α-synuclein containing one SUE1 tag and one LACE tag and 16 μM K48- linked diUb were mixed and reacted in reaction buffer to achieve almost complete ubiquitination of α-synuclein (typically 2 h), and then 1 μM chimera E1 V4.5, 40 μM UBC9 K14R and 160 μM monoUb were added to the reaction buffer at 37 °C for another 2 h. These reactions were sampled and an equal volume of 4x sodium dodecyl sulfate (SDS) sample buffer containing 400 mM DL-dithiothreitol (DTT) was added and then analyzed by SDS‒PAGE, imaged by fluorescence.