Pig tail

GFP-binding protein (GBP; Rothbauer et al., 2008 (link)) was fused to the Fc domain of human IgG (pIg-Tail; R&D Systems), tagged with His₆ in pET28a (EMD Biosciences), expressed in Escherichia coli, and purified on HisPur resin (ThermoFisher; catalogue no. 88221) as described previously (Buster et al., 2013 (link)). Purified GBP was bound to magnetic Dyna Beads (ThermoFisher; catalogue no. 10001D), and then cross-linked to the resin by incubating with 20 mM dimethyl pimelimidate dihydrochloride in PBS, pH 8.3, 2 h at 22°C, then quenched by incubation with 0.2 M ethanolamine, pH 8.3, 1 h at 22°C. Antibody-coated beads were washed three times with PBS-Tween20 (0.02%), then equilibrated in 1.0 ml of cell lysis buffer (CLB; 50 mM Tris, pH 7.2, 125 mM NaCl, 2 mM DTT, 0.1% Triton X-100, and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]). Transfected cells expressing recombinant proteins were lysed in CLB, and the lysates clarified by centrifugation at 16,100 × g for 5 min at 4°C. Inputs (0.5–1%) were used for immunoblots. GBP-coated beads were rocked with lysate for 30 min at 4°C, washed three times with 1 ml CLB, and then boiled in Laemmli sample buffer.

GFP-binding protein (GBP; Rothbauer et al., 2008 (link)) was fused to the Fc domain of human IgG (pIg-Tail; R&D Systems), tagged with His₆ in pET28a (EMD Biosciences), expressed in Escherichia coli, and purified on HisPur resin (Thermo Fisher Scientific) according to the manufacturer’s instructions (Buster et al., 2013 (link)). Purified GBP was bound to magnetic Dyna Beads (Thermo Fisher Scientific) and cross-linked to the resin by incubating with 20 mM dimethyl pimelimidate dihydrochloride in PBS, pH 8.3, for 2 h at 22°C and then quenching the coupling reaction by incubating with 0.2 M ethanolamine, pH 8.3, for 1 h at 22°C. Antibody-coated beads were washed three times with PBS–Tween 20 (0.02%) then equilibrated in 1.0 ml cell lysis buffer (CLB; 50 mM Tris, pH 7.2, 125 mM NaCl, 2 mM DTT, 0.1% Triton X-100, 1× protease inhibitor cocktail [Roche], and 0.1 mM PMSF) or CLB containing phosphatase inhibitors for phospho-specific antibody detection (CLB+; CLB with 200 mM NaF, 150 mM β-glycerophosphate, and 1 mM Na₃VO₄). Transfected cells expressing recombinant proteins were lysed in CLB or CLB+, and the lysates were clarified by centrifugation at 16,100 g for 5 min at 4°C. 0.5–1% of the inputs were used for immunoblots. GBP-coated beads were rocked with lysate for 30 min at 4°C or 10 min at 22°C, washed four times with 1 ml CLB or CLB+, and boiled in Laemmli sample buffer.