Polyubiquitin-B

The semisynthetic ubiquitination reaction was performed without major modifications to the already outlined protocol. Briefly, Ub-SH was activated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and incubated with each tau mutant in a 1:4 tau:Ub molar ratio. The desired proteoform was then purified through ion exchange chromatography exploiting a linear gradient of NaCl.