Generating Polyubiquitin-UBB+1 Conjugates

Conventional linkage specific enzymes were used to generate polyUb–UBB⁺¹. By default UBB⁺¹ served as the proximal unit, while the chain length was controlled by addition of Ub^K48R/K63R for dimeric conjugates or Ub^WT for longer forms (17 (link), 18 (link)). In brief NLEL^170-782/UbcH7, UBE2S, E2-25K, and Ubc13/Uev1 were used to make K6, K11, K48, and K63 linkages, respectively according to (19 (link), 20 (link)). Incorporation of an N-terminal 6xHis tag on UBB⁺¹ allowed for easy isolation of polyUb–UBB⁺¹ conjugates, which were then separated on a superdex 75 16/60 size exclusion column (GE Life Sciences). Immunoblots of conjugates were performed with several rabbit primary antibodies in a 1:1,000 dilution: anti-His₆ (GenScript A00174-40), anti-ubiquitin (Dako Z0458), anti-K48 ubiquitin clone Apu2 (Millipore 05-1307), anti-K63 ubiquitin clone Apu3 (Millipore 05-1308). All polyUb conjugates were run on 4–20% SDS-PAGE (GeneScript) and transferred to a PVDF membrane (Thermo Scientific) using an eblot Protein transfer system (Genscript). Membranes were incubated with IgG goat anti-rabbit HRP (Bio-Rad) secondary antibody in a 1:50,000 dilution and visualized using a ChemiDoc Imaging system (BioRad). For unambiguous communication, UBB⁺¹ conjugates are named following the system from (21 (link)) throughout.