Rabbit anti k48 ub clone apu2

RING stimulation of UBE2K (Fig. 1a and Extended Data Fig. 1d) was carried out in assay buffer (25 mM Tris–HCl, 50 mM NaCl, pH 8.0) with 15 mM ATP, 15 mM MgCl₂, 1 μM UBA1, 200 μM Ub^WT and 5 μM UBE2K at 37 °C. The concentration of E3s was kept at 5 μM. UBE2K D124 variants (Fig. 1b) were assessed under identical conditions. Reactions were stopped at indicated time points with LDS loading buffer and resolved by SDS–PAGE. Gels were transferred to nitrocellulose membrane (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad) for 10 min with constant 1.3 amps. Total protein was visualized with Ponceau-S, imaged using Bio-Rad ChemiDoc, destained and the membrane was blocked in 5% (w/v) BSA in TBST (20 mM Tris–HCl, 150 mM NaCl, 0.1%(w/v) Tween-20) for 30 min at ambient temperature. Primary antibodies were incubated at 4 °C for 12 hours using a 1:5,000 dilution of mouse anti-Ub (PD41, Santa Cruz Biotechnology) and 1:3,000 dilution of rabbit anti-K48-Ub (clone Apu2, Merck) in 2.5% (w/v) BSA in TBST. The membrane was washed three times for 5 min in TBST and incubated with secondary antibodies: IRDye 800CW goat anti-mouse IgG (LI-COR Biosciences) in a 1:15,000 dilution and IRDye 680RD goat anti-rabbit IgG (LI-COR Biosciences) in a 1:15,000 dilution, for 1 hour at ambient temperature. Two washes with TBST and a final in TBS were carried out before imaging on a LI-COR Odyssey CLx.