Western Blot for Protein Quantification

Western blot was performed as described (3 (link),37 (link)). Briefly, 0.1 OD₆₀₀ bacterial culture was collected by centrifugation for 2 min at 8000 rpm at 4°C, and resuspended in 100 μl 1× protein loading buffer. After heating for 5 min at 95°C, 0.02 OD₆₀₀ of cell lysate was separated on a 10% SDS-PAGE gel. Proteins were transferred onto a NC membrane (#10600002, GE Healthcare) for 60 min at 100 V in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, pH 8.3). Membranes were blocked for 1 h at room temperature in 1× TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween20, pH 7.6) buffer with 5% (w/v) Difco™ skim milk (#6307915, BD). After blocking, membranes were incubated at room temperature for 1 h with monoclonal α-FLAG (Sigma-Aldrich #F1804; 1:1000) or polyclonal α-GroEL (Sigma-Aldrich #G6532; 1:5000) antibodies diluted in 1× TBST buffer containing 3% BSA. Following three washes for 30 min in 1× TBST buffer, membranes were incubated with secondary α-mouse or α-rabbit HRP-linked antibodies (Sigma-Aldrich #A0168 or #A0545; 1:5000) diluted in 1× TBST buffer containing 3% BSA. After another three washes for 30 min in 1× TBST buffer, chemiluminescence was developed using the Novex™ ECL Chemiluminescent Substrate Reagent Kit (#WP20005, Thermo Fisher Scientific), visualized on ChemiDocTM XRS+, and quantified using ImageLabTM Software (both Biorad).