Gfp trap coupled agarose beads

Gim3-TAP yeast cells transformed with a control empty vector (BPM 42), P_GPD-GFP (BPM 779), P_GPD-Guk1-GFP (BPM 780), or P_GPD-Guk1-7-GFP (BPM 781) were grown to log phase and then lysed with glass beads and native lysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X protease inhibitor mix, 1 mM 1,10 phenanthroline, 1 mM EDTA, 10 mM iodoacetamide). To pulldown GFP-tagged proteins, lysates were incubated for 2 hours at 4°C with 20 μL GFP-Trap coupled agarose beads (Chromotek). Beads were washed three times in lysis buffer before samples were eluted with 3X SDS sample buffer. Nitrocellulose membranes were probed with mouse anti-GFP (Roche, 1:2,500), rabbit anti-Pgk1 (Acris Antibodies, 1:10,000), rabbit anti-TAP (Fisher, 1:2,500), and mouse anti-ubiquitin (Millipore, 1:2,500) primary antibodies.

Approximately 100 OD₆₀₀ of log-phase cells grown exponentially in YPD were harvested and washed twice with water then lysed by beating with 0.5-mm-diameter glass beads (Sigma-Aldrich) and ice cold, freshly prepared native lysis buffer (20 mM HEPES, pH 7.5, 0.5% Triton X-100, 200 mM NaCl, 1X protease inhibitor mix, 1 mM 1,10 phenanthroline, 1 mM EDTA, 10 mM iodoacetamide). To pulldown GFP-tagged proteins, lysates were incubated for 2 hours at 4°C with 25 μL GFP-Trap coupled agarose beads (Chromotek). After incubation, the agarose beads were washed three times in lysis buffer before samples were eluted with 2X SDS sample buffer. Nitrocellulose membranes were probed with rat anti-GFP (3H9, Chromotek, 1:2,500), PAP (Sigma-Aldrich, 1: 2,500), and mouse anti-HA (Y-11, Santa Cruz Biotechnology, 1:2,500) primary antibodies.

Cells expressing ectopic Guk1-7-GFP, Guk1-GFP, or a control empty vector (pRS313) were grown to log phase and then lysed with glass beads in native lysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 200 mM NaCl, 1X protease inhibitor mix (Roche), 1 mM 1,10 phenanthroline, 1 mM EDTA, 10 mM iodoacetamide). GFP-tagged Guk1-7 was pulled down with GFP-Trap coupled agarose beads (Chromotek; 10 µL per 3 mg of lysate) for 2 hours at 4°C. Beads were washed three times in lysis buffer before samples were eluted with 3X SDS buffer. Equal volumes of samples were resolved by SDS-PAGE. Membranes were immunoblotted with mouse anti-GFP (Roche, 1:2,500), rabbit anti-Pgk1 (Acris Antibodies, 1:10,000), and mouse anti-ubiquitin (Millipore, 1:2,500) primary antibodies and secondary antibodies (Mandel Scientific, 1:10,000).