Figure 1.

Large-scale production of monoubiquitinated proteins. (A) In vitro ubiquitination reaction (Step 1) to produce ubiquitinated products with native isopeptide linkages, highlighted in the dashed square (Step 2). The lower panel denotes the constructs that were used (Figure S1 and Table S1). The reaction components were subjected to affinity chromatography (Steps 3 and 4) for a selective purification of monoubiquitinated products. Western blot and SDS-PAGE analyses of corresponding reactions of (B) αSyn_1–140^His + NEDD4L and (C) ALIX_1–868* + WWP2; 4–12% Bis-Tris and 3–8% Tris-Acetate gels were used for αSyn_1–140^His and ALIX_1–868*, respectively. Aliquots from each step are designated by a circled number.