Mini edta free protease inhibitor tablet

Cells cultured on 10 cm plates were lysed in 500 μl of lysis buffer: 10 mM Tris-HCL pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 0.5% Nonidet P-40, 1 mM PMSF and mini EDTA- free protease inhibitor tablet (Roche, Basel, Switzerland) according to the manufacturer’s protocol. 100 μl of lysate was stored as a whole cell lysate control and heated with 5x Laemmli sample buffer (LSB) for 5 min at + 95 °C. After centrifugation at 500 x g for 5 min at + 4 °C, the supernatant contained the cytoplasm, while the nuclei were in the pellet. The pellets were washed three times with lysis buffer and centrifuged each time at 500 x g for 5 min at + 4 °C, after which they were suspended to 200 μl of lysis buffer. The cytoplasm-containing solution was centrifuged at 12000 x g for 15 min at + 4 °C, after which the supernatant was collected. Nuclear and cytoplasmic lysates were heated for 5 min at + 95 °C with 5x LSB. 30 μl aliquots of samples were loaded to each well on SDS-PAGE and samples were analysed by Western blotting, using nuclear A/C laminin and cytoplasmic beta-tubulin as fraction-specific positive controls (Additional file 1: Table S4).

His-tagged S. aureus RnpA was purified as previously described [14 (link)]. Briefly, E. coli BL21 (DE3) cells harboring plasmid pEXP5-nt [36 (link)], containing a hexahistadine tag fused to the N-terminus of the S. aureus RnpA coding region under the control of the plasmid’s T7 promoter, were cultured to an OD₆₀₀ of approximately 0.6 and then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for three hours to induce protein production. E. coli cells were collected by centrifugation at 9000 rpm for 10 min at 4 °C and then suspended in 20 mL of buffer A (300 mM NaCl, 50 mM Na₂HPO₄, pH 7.4) containing a mini EDTA-free protease inhibitor tablet (Roche; Branford, CT, USA) and 20 mM imidazole. Cells were mechanically ruptured by three passes at 18,000 psi through a French Pressure Cell Press (SLM-Aminco; Pittsford, NY, USA), and cell debris was removed by centrifugation at 4 °C at 17,000 × g for 10 min at 4 °C. Supernatants were collected, filtered through a 0.2 μm syringe filter then loaded onto the BioRad Maximizer Duo-Flow Medium Pressure Chromatography System containing a 5 mL HisPur Cobalt Column (Thermo Scientific). Protein was eluted using an imidazole gradient (80 mM to 500 mM); fractions were assessed for RnpA presence and purity on SDS-PAGE gels via Coomassie staining and Western blotting using anti-His antibody (Invitrogen).

His-tagged RnpA was purified as previously described [33 (link)]. E. coli BL21 (DE3) cells harboring plasmid pEXP5-nt containing a hexahistidine tag fused to the N-terminus of S.aureus RnpA coding region were grown in LB supplemented with 50 µg mL⁻¹ ampicillin to OD₆₀₀ = 0.6 and induced with 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for three hours to induce protein expression. E. coli cells were subjected to centrifugation at 1560× g for 20 min at 4 °C and the cell pellet was resuspended in 20 mL buffer A (300 mM NaCl, 50 mM Na₂HPO₄, pH 7.4) containing a mini EDTA-free protease inhibitor tablet (Roche; Bradford, CT, USA) and 20 mM imidazole. Cells were mechanically lysed by three passes at 18,000 psi through a French Pressure Cell Press (SLM-Aminco; Pittsford, NY, USA) and cell debris was removed by centrifugation at 17,000× g for 10 min at 4 °C. Supernatants were collected, filtered through a 0.2 micron syringe filter, then loaded onto a 5 mL HisPur cobalt column (ThermoFisher Scientific, Waltham, MA, U.S.A.) using the BioRad Maximizer Duo-Flow Medium Pressure Chromatography System. Protein was eluted using an imidazole gradient (80 mM to 500 mM). Fractions were assessed for RnpA presence and purity via SDS-PAGE analysis, Coomassie staining and Western blotting using an anti-His antibody (Invitrogen; Grand Island, NY, USA).