Ni sepharose

E. coli BL21 cells expressing His₆-tagged Pol30 or GST-tagged Srs2-CT Rad30-PIP were induced by 0.1 mM IPTG at 16°C for 16 to 18 h. Cells were harvested by centrifugation, resuspended in corresponding His₆ lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 30 mM imidazole) or GST lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 10 mM β-mercaptoethanol) and then homogenized in a cell disruptor (Constant Cell Disruption Systems, CF1) at 25 lb/in² by two passes. Cells lysates were centrifuged at 16,000 g for 60 min. His₆-Pol30 was affinity purified by Ni Sepharose (Cytiva, 17531801) and then eluted from the Ni Sepharose by 5-fold bed volume elution buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 500 mM imidazole, 20% glycerol). GST-Srs2-CT and GST-Rad30-PIP were affinity purified by glutathione Sepharose (Cytiva, GE17-0756-01) in a GST stock buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 10 mM β-mercaptoethanol, 20% glycerol). The above proteins were either freshly used or frozen in liquid nitrogen and stored at −80°C. Equal molars of GST-Srs2/Rad30 and His₆-Pol30 were coincubated overnight at 4°C with gentle shaking. Anti-GST (Sigma, G7781-25UL, 1:1000), anti-His₆ (New England Biolabs, 12698, 1:1000) primary antibodies and the anti-Rabbit (Bio-Rad, 1706515, 1: 3000) secondary antibody were used in immunoblotting.

Constructed Gal4_AD and Gal4_BD vectors were transformed into the yeast two-hybrid strain PJ69-4a (James et al., 1996 (link)) in pairs and allowed to grow at 30°C for 2–3 days. Transformants were selected on SD-Leu-Trp plates. Protein interaction was determined on synthetic complete medium lacking Trp, Leu and His, supplemented with 3-amino-1,2,4-triazole (3-AT, Sigma-Aldrich), or on plates lacking Trp, Leu, and Ade. SD-Leu-Trp plates were used as a control.
Full-length BdUEV1 ORFs were cloned in plasmid pGEX-6p and BdUBC13s were cloned in plasmid pET30a as previously described (Guo et al., 2016 (link)). The His₆ and GST fusion constructs were transformed into E. coli strain BL21 (DE3) and the recombinant proteins were purified with Ni Sepharose and glutathione (Amersham Pharmacia), respectively. For the pull-down assay, crude cell extracts were loaded on glutathione Sepharose^TM 4B beads and then 10 μg of purified His₆-BdUbc13 was later added. After incubation and washing, the GST beads were boiled with SDS-PAGE loading buffer for 10 min prior to Western blotting.

Full-length BdUBC13A and BdUBC13B were cloned in plasmid pET30a(+). Full-length AtUEV1A, AtUEV1B, AtUEV1C, and AtUEV1D were cloned in plasmid pGEX-6p-1. The His₆ and GST fusion constructs were transformed into E. coli strain BL21 (DE3). The recombinant proteins were purified with Ni Sepharose and glutathione (Amersham Pharmacia) according to the manufacturer’s protocol.
For the pull-down assay, crude cell extracts were loaded on glutathione Sepharose^TM 4B beads and then 10 μg of purified His₆-BdUbc13A or His₆-BdUbc13B was later added. After incubation and washing, the GST beads were boiled with SDS-PAGE loading buffer for 10 min before western blotting analysis.