Bolt bis tris sds page gels

M1-linked Ub chain formation assays used to probe the overall HOIP E3 ligase activity were performed as previously described^{6 (link)}. 200 nM E1, 1 µM E2 (UbcH5B), 1 µM HOIP RBR and 40 µM Ub with native N- and C-termini were incubated in 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM MgCl₂, 0.6 mM DTT, and 10 mM ATP in the presence or absence of 10 µM fully blocked M1-linked di-Ub or fully blocked I44A M1-linked di-Ub at 30 °C. Reactions were stopped at different time points by adding samples to 2x SDS-sample buffer with DTT and heating for 5 min to 95 °C. Samples were analysed on 4–12% Bolt Bis-Tris SDS-PAGE gels (Invitrogen) and visualized using Coomassie Brilliant blue dye.

The HOIP-Ub thioester formation assay used to probe the first step of the HOIP RBR reaction is based on previously described methods^{6 (link)}. 100 nM E1, 2 µM E2 (UbcH5B), and 8 µM Ub were incubated in 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM MgCl₂, and 5 mM ATP for 5 min at 25 °C to pre-load E2 with Ub. HOIP RBR was preincubated with blocked di-Ub (GPG-di-Ub∆C-term Gly) for 5 min at 25 °C and added to the loaded E2-Ub mixture to final concentrations of 1 µM HOIP RBR. Reactions were stopped after 30 s by mixing samples 1:1 v/v with pre-heated 2x SDS loading buffer without DTT, analysed on 4–12% Bolt Bis-Tris SDS-PAGE gels (Invitrogen) and visualized using Coomassie Brilliant blue dye. Gels were scanned on a Li-COR Odyssey scanner using the 700 nm channel and bands quantified using ImageStudio software (LI-COR). The amount of HOIP-Ub thioester was determined as the fraction of HOIP-Ub to total HOIP for each sample. Data were plotted and analysed in GraphPad Prism.