Protein Extraction and Precipitation Protocol

The protocol reported by Niu et al. [12 (link)] was used for protein extraction and precipitation with some modifications. The supernatants were precipitated for 6 hours by adding 5 ml of ice-cold trichloroacetic acid (TCA)/acetone buffer (10% TCA (Merck) and 10 mM dithiothreitol (DTT)(Sigma-Aldrich) in acetone (Fisher Scientific)) to 1 ml of the sample. Then, the sample was centrifuged at 15,000 g for 10 min at 4°C, and the supernatant was discarded. The precipitated pellet was washed thrice with ice-cold 10 mM DTT (Sigma-Aldrich)-acetone (Fisher Scientific) solution and air-dried in a fume hood for 20–30 minutes. The protein pellet was resuspended in 300 μl of solubilization buffer [8 M urea (Sigma-Aldrich), 10 mM DTT (Sigma-Aldrich), 0.1 M triethylammonium bicarbonate (TEAB)(Sigma-Aldrich) at pH 8.5] by vortexing for 20 minutes. The sample was then centrifuged at 15,000 g for 15 min at 20°C twice, and the pellet was discarded.