Mini edta free protease inhibitor cocktail

Strain BL21DE3/pET16b-His-CsrA, a generous gift from Charles Midgett, was grown to an OD₆₅₀ of approximately 0.6 at 37°C with shaking. CsrA production was induced by the addition of 500 μM IPTG (isopropyl-β-d-thiogalactopyranoside) for 4 h at 23°C with shaking. The cells were centrifuged at 5,500 × g for 10 min at 4°C, washed with 50 mM NaCl, and stored at −80°C. The cells were lysed by resuspending the cells in 10 ml of lysis buffer (50 mM NaH₂PO₄, 200 mM NaCl, 10 mM imidazole, 1% Triton X-100, 10% glycerol, and one tablet of cOmplete, Mini, EDTA-free protease inhibitor cocktail (Sigma-Aldrich) followed by four rounds of 30 s of sonication, each followed by 30 s on ice. Cell debris was pelleted by centrifugation at 15,000 × g at 4°C. The supernatant was incubated with 5 ml of nickel agarose beads for 3 h with rotation. The slurry was washed with 50 ml of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, and 0.1% Triton X-100). Elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 500 mM imidazole) was used to elute His-TEV-CsrA in 1-ml fractions. Relevant factions were pooled, tobacco etch virus (TEV) protease was added at a 1:10 (TEV/CsrA) ratio, and the reaction mixture was incubated overnight at 4°C. The reaction mixture was then passed through a nickel agarose column, and purified CsrA was concentrated and stored in storage buffer (50 mM KPO₄, 50 mM NaCl, 50% glycerol).

For protein detection, 25,000 cells/cm² were seeded in a 6-well plate and exposed to NPs at the indicated concentrations for 24 h. Following exposure, cells were collected and lysed overnight at 4°C in RIPA buffer [50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM EDTA]. Protease- and phosphatase inhibitors (Mini EDTA-free Protease Inhibitor Cocktail, Sigma Aldrich; 1 mM PMSF, Thermo Fisher; PhosSTOP™, Sigma Aldrich) and 1 mM DTT (Sigma Aldrich) were freshly added to the buffer. Cell lysates were centrifuged at 13.000 × g for 15 min and supernatants were collected. The protein concentration was measured using the Bradford assay and 30 µg were loaded into each well of a NuPAGE 4%–12% Bis-Tris gradient gel (Thermo Fisher). Following electrophoretic separation, the proteins were transferred to a Hybond Low-fluorescent 0.2 µm PVDF membrane (Amersham), blocked for 1 h in Odyssey^® Blocking Buffer (PBS) (LI-COR), and stained overnight at 4°C with primary antibodies against NRF2 (Abcam, ab62352). Antibodies against β-actin (Sigma-Aldrich) were used for loading control, and the goat anti-mouse IRDye 680RD antibody (LI-COR Biotechnology GmbH, Bad Homburg, Germany) was used as a secondary antibody. Proteins were detected using LI-COR Odyssey^® CLx scanner and Odyssey^® Image Studio software.