Irdye 680lt goat anti mouse igg h l secondary antibodies

B. burgdorferi clones were grown to late stationary phase (24 h after reaching stationary, 2.5–3 × 10⁸ cells/ml) in BSK-II medium. Spirochetes were harvested by centrifugation at 3210g for 10 min at 20°C and washed twice in 1 ml of cold HN buffer (50 mM Hepes, 50 mM NaCl, pH 7.4). Cell pellets were resuspended in protein sample buffer to a final density of 1 × 10⁶ cells/μl. Total protein lysates (1 × 10⁷cells/lane) were separated by SDS-PAGE and transferred to a PVDF membrane. Immunoblots were performed using both rabbit anti-GlpD (1:5000) (Drecktrah et al., 2022 (link)) and mouse anti-FlaB H9724 (1:200) (Barbour et al., 1986 (link)) or rabbit anti-Rrp1 (1:1000) (Bontemps-Gallo et al., 2016 (link)) and anti-FlaB, in a 1:1 solution of Odyssey blocking buffer (LI-COR Biosciences) and Tris-buffered saline, pH 7.4 and 0.1% Tween20 (TBST) followed by IRDye 800CW goat anti-rabbit IgG and IRDye 680LT goat anti-mouse IgG (H + L) secondary antibodies (LI-COR Biosciences). Immunoblots were visualized and quantified using the LI-COR Odyssey scanner and software (Image Studio version 4). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple-comparisons test to wild-type (GraphPad Prism, version 9.0.0).