Figure 2.

Chemical assembly of K11-linked di- and tri-Ub chains. (a,c,e) Coomassie-stained 15% SDS PAGE gels of the chemical condensation reactions of (a) K11-linked Ub₂ ¹⁵N-labeled on the proximal Ub, (c) K11-linked Ub₃ with the middle Ub unit ¹⁵N-labeled, and (e) K33,K11-linked Ub₃ with the middle Ub unit ¹⁵N-labeled. K11-linked Ub₂ (panel a) was assembled from Alloc-protected monomers of Ub-SR (Ub-SR^A) and TFA-treated ¹⁵N-labeled K11Boc Ub (TFA K11B Ub^A). K11-linked Ub₃ (panel c) was assembled from Alloc-protected Ub-SR and distal-¹⁵N-labeled K11-linked Ub₂ (TFA K11B Ub₂^A) whose distal-K11 side chain was made available for ligation by TFA treatment. K33,K11-linked Ub₃ (panel e) was assembled from Alloc-protected proximal-¹⁵N- labeled K33-linked Ub₂ (K33-Ub₂-SR) and TFA-treated K11Boc Ub (TFA K11B Ub^A). Formation of Ub₂ or Ub₃ is observed after 20 hours of reacting the proteins with AgNO₃, H-OSu, and DIEA. 0.3 – 0.4 μL are loaded from the reaction directly onto gel. Notations used here: “TFA” indicates that the corresponding protein was treated with TFA to remove the Boc group in order to make the corresponding Lys available for the ligation reaction, while the superscript “A” indicates that the protein is Alloc-protected.

(b,d,f) Size-exclusion chromatograms of (b) K11-linked Ub₂, (d) homogeneously K11-linked Ub₃, and (f) mixed-linkage K33,K11-linked Ub₃ performed after Alloc deprotection and protein renaturation to purify the desired product from the unreacted components. The ability to separate Ub₂ or Ub₃ products from unreacted species is illustrated in Fig. S8.