Sigmafast protease inhibitor cocktail tablet

GFP IPs were described before^{21 (link)}. For RCP/ P-gp IPs, cells from each 10 cm tissue culture dish were lysed by scrapping in 200 µL of NDLB (5 M NaCl, 1 M Tris-HCl, pH 7.5, 0.5 M EDTA, 0.5 M EGTA, 0.5 M NaF, 0.1 M NaVO4) and supplemented with complete protease inhibitor tablet, 0.15% Tween 20 and 10% SIGMAFAST™ Protease Inhibitor Cocktail Tablet (Sigma). Scraped cells were passed through a 27.5 G needle (5X) and centrifuged at maximum speed for 10 min at 4 °C. For each protein lysate, 40 µL of the sheep-anti-mouse IgG dynabeads (Thermo scientific) or 40 µL of the Dynabeads™ Protein G (Thermo scientific) were coupled with antibodies against P-gp (8 µg), GFP (0.75 µg, Ab6556, Abcam) or RCP (4 µL, D9D8P, Cell Signalling) for 1 h at 4 °C and antibody coupled beads were further incubated with the protein lysates overnight at 4 °C under constant rotation. Unbound proteins were washed by 4X washing with the NDLB lysis buffer and eluted in 40 µL of 2X reducing sample buffer and boiled for 10 min. One example is shown, but verified independently in two additional repeats.

The alcohol dehydrogenase activity in cell free extracts was assayed by washing 2 mL of a liquid overnight culture on YPD at 25°C with 1 mL PBS and resuspending it in 500 µL cell lysis buffer with glass beads. The modified lysis buffer consisted of 20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl₂, 10% glycerol, 1 SIGMAFAST™ Protease Inhibitor Cocktail Tablet per 50 mL (Sigma-Aldrich GmbH) (Karaoglan, Karaoglan and Inan 2015 (link)). The cultures were lysed by bead beating (FastPrep-24, MP Biomedicals, Inc.) for 3 × 20 s at 6 m s^–1 with 1-minute cooling on ice in-between steps. After the lysis step, the cultures were centrifuged, and the supernatant was transferred to a fresh microcentrifuge tube and centrifuged again at 13 200 rpm for 30 min at 4°C to remove any carried over cell debris. After the second centrifugation step the supernatant was stored at −20°C till use.

Crude membranes of yeast were prepared by disrupting the cells mechanically with glass beads, as described previously [37 (link)]. Briefly, cells were resuspended in lysis buffer containing 20 mM Tris-HCl (pH = 7.5), 500 mM NaCl, and 10% v/v glycerol and protease inhibitors (1 mM PMSF and 1 SIGMAFAST protease inhibitor cocktail tablet (Sigma, St. Louis Missouri, MO, USA) at a ratio of 40–50 g wet cell weight for 200 mL of buffer. Cells were lysed through high-speed mixing in the presence of glass beads for 6 × 1 min, with 2 min breaks on ice. The supernatant was collected and the glass beads were washed twice in 50 mL of ice-cold lysis buffer. Unbroken cells and cell debris were removed from the combined supernatants by centrifugation at 5000× g for 20 min at 4 °C. The membranes were pelleted via ultracentrifugation at ~142,000× g for 3 h at 4 °C. The membranes were resuspended in lysis buffer using a ratio of 1 g of wet weight of membrane per 50 mL of buffer with protease inhibitors, and kept at −80 °C until further usage.

The procedure for viral minichromosome enrichment has been previously described [18 (link)]. Briefly, 2 g of fresh tissue was ground in liquid nitrogen until a fine powder was obtained. The powder was then homogenized at 4 °C in 20 mL of 10mM Tris-HCl buffer, pH 9.0 that contained 500 mM sucrose, 80 mM KCl, 0.5 mM spermidine, 0.5 mM spermine, 0.5% Triton X-100, 10mM EDTA, and 15 mM β-mercaptoethanol supplemented with Sigmafast protease inhibitor cocktail tablet (Sigma-Aldrich, St. Louis, MO, USA). The resulting homogenate was first filtered through two layers of cheesecloth and, then, two layers of Miracloth (475855; Calbiochem). The filtrate was centrifuged for 15 min at 2000× g (e.g., Sorval RC-5B, ss-34 rotor). The nuclei-rich sediment was resuspended in 0.5 mL of extraction buffer (10 mM Tris-HCl pH 8 plus 0.1% Sarkosyl detergent) and immediately incubated on ice for 15 min. The suspension was then centrifugated at 2000× g for 15 min. The supernatant (nuclear extract) containing viral minichromosomes was recovered and saved for further analysis.

HerA and NurA cells were re-suspended in lysis buffer (100 mM NaCl, 50 mM Hepes–NaOH pH 8.0, 5 mM DTT and 5% v/v glycerol) supplemented with an EDTA-Free SigmaFast Protease Inhibitor Cocktail Tablet (Sigma-Aldrich). The cells were lysed by sonication and the lysate was clarified by centrifugation. The supernatant was heated in a 70°C water bath for 20 min and subsequently clarified by centrifugation to remove precipitated protein. The supernatant was then loaded onto a 5 ml Heparin HP column (GE Healthcare) equilibrated with lysis buffer. Bound protein was eluted with a linear gradient from 100 mM to 1 M NaCl. Fractions containing the protein of interest were pooled and dialysed against 100 mM NaCl, 50 mM Hepes–NaOH pH 8.0, 5 mM DTT and 5% (v/v) glycerol for 18 h. The dialyzed protein was loaded onto a 5 mL HiTrap Q HP column (GE Healthcare) equilibrated with lysis buffer. Bound protein was eluted with a linear gradient from 100 mM to 1 M NaCl. Fractions containing the protein of interest were pooled and dialysed against 300 mM NaCl, 20 mM Hepes–NaOH pH 8.0, 5 mM DTT and 5% (v/v) glycerol for 18 h. The protein was concentrated with Amicon Ultra-15 centrifugal filter units (EMD Millipore) and aliquots were flash-frozen in liquid nitrogen and stored at –80°C. Protein concentrations were estimated from absorbance at 280 nm using extinction coefficients.