Goat anti mouse lgg hrp secondary antibody

Spirochetes were harvested by centrifugation at 8000×g for 10 min and washed three times with PBS (pH 7.4) at 4 °C. Pellets were suspended in SDS buffer containing 50 mM Tris-HCl (pH 8), 0.3% sodium dodecyl sulfate (SDS), and 10 mM dithiothreitol (DTT). Cell lysates (5 × 10⁷ cells per lane) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (GE-Healthcare, Milwaukee, WI). Membranes were blotted with antibodies against FlaB (monoclonal, 1:1000 dilution), LtpA (polyclonal, 1:2000 dilution)^{30 (link),41 (link)}, and then incubated with goat anti-mouse lgG-HRP secondary antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-rat lgG-HRP secondary antibody (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA). The detection of horse radish peroxidase activity was determined using the enhanced chemiluminescence method (Thermo Pierce ECL Western Blotting Substrate, Waltham, MA) and subsequently by exposure to X-ray film.

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.

Spirochetes from mid-log cultures were harvested by centrifuging at 8,000 × g for 10 min and followed by three time washing with PBS (pH 7.4) at 4°C. Pellets were suspended in SDS buffer containing 50 mM Tris-HCl (pH 8.0), 0.3% sodium dodecyl sulfate (SDS) and 10 mM dithiothreitol (DTT). Cell lysates (10⁸ cells per lane) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (GE-Healthcare, Milwaukee, WI). Membranes were blotted with mouse polyclonal antibody against VlsE (1:1,000 dilution) [76 ] and monoclonal antibody against FlaB (1:1,000 dilution) [41 (link)], and then incubated with goat anti-mouse lgG-HRP secondary antibody (1:1,000; Santa Cruz Biotechnology). Detection of horseradish peroxidase activity was determined by the enhanced chemiluminescence method (Thermo Pierce ECL Western Blotting Substrate) with subsequent exposure to X-ray film.