Ups2 spike in

From each fraction, 90 µl was mixing with 30 µl of 5x Laemmli loading buffer. The fractions were analyzed in 12% SDS-Page standard gel followed by Coomassie staining (20 µl for the fractions, 3 µl for the lysate and 10 µl for the pellet). KhpB was detected by the αKhpB antibody (2, SY0917 (MA11010) SABC-serum) (rabbit, 1:10,000) and a secondary anti-rabbit-HRP (goat) conjugate antibody ( 1:10,000, Sigma-Aldrich, #31460). The rest of the samples was prepared for mass spectrometric analysis as previously described (Hör et al. 2020b (link)). Briefly, samples were homogenized using ultrasound. Debris were later removed by centrifugation and 20 µl of the cleared protein sample were spiked-in with 10 µl of UPS2 spike-in (Sigma-Aldrich) diluted in 250 µl of 1.25x protein loading buffer. The samples were then reduced in 50 mM DTT, 10 min at 70°C and alkylated with 120 mM iodoacetamide for 20 min at room temperature in the dark. The proteins were precipitated using four times their volume of acetone overnight at −20°C, and later on washed with acetone and dissolved in 50 µl of 8 M urea, 100 mM ammonium bicarbonate. Protein digestion into peptides was performed using Lys-C (Wako) for 2 h at 30°C following by overnight digestion by trypsin. Peptides were eluted with 60% acetonitrile/0.3% formic acid and stored at −20°C until LC-MS7MS analysis.