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Two Ub-K48R modifications can be added onto Ub-Ub-GFP. Time course of reaction of 10 µM Ub-Ub-GFP with 200 µM Ub-K48R under conditions identical to those used to make substrate produced pure Ub1Ub-Ub1Ub-GFP, indicating that ubiquitin chains are assembled on both fused ubiquitins in Ub-Ub-GFP. SDS/PAGE analysis of unboiled samples. GFP was detected by excitation at 488 nm.
