SDS-PAGE Analysis of Borrelia Proteins

The method for SDS-PAGE was described previously (Carrasco et al., 2015b (link)). Briefly, spirochetes were cultured from 10⁴ cells/ml and harvested at day 7 (stationary phase 10⁸ cells/ml) by centrifugation at 8,000 g for 10 min and washed two times with PBS (pH 7.4) at 4°C. Pellets were suspended in SDS buffer containing 50 mM Tris-HCl (pH 8.0), 2% sodium dodecyl sulfate (SDS), and 10 mM dithiothreitol (DTT). Cell lysates (5 × 10⁷ cells per lane) were separated by 12% SDS-PAGE and stained with Coomassie blue or transferred to nitrocellulose membranes (GE-Healthcare, Milwaukee, WI). Membranes were blotted with monoclonal antibodies against FlaB, RpoS, and BosR (He et al., 2008 (link); Xu et al., 2010 (link); Troxell et al., 2013 (link)) with 1:1,000, 1:50, and 1:500 dilutions, respectively, and then with goat anti-mouse lgG-HRP secondary antibody (1:1,000, Santa Cruz Biotechnology). Detection of horseradish peroxidase activity was determined using enhanced chemiluminescence method (Thermo pierce ECL Western Blotting Substrate), and subsequently by exposure to X-ray film.