Borrelia Species Protein Extraction

Low-passaged (≤10 laboratory passages) cultures of B. burgdorferi B31 A3 (63 (link)), B. turicatae 91E135 (64 (link)), B. parkeri SLO, and B. hermsii HCT-4 (29 (link)) were grown at 35 to 37°C to densities of  >1 × 10⁷ cells mL⁻¹ in 40 mL modified Barbour-Stonner-Kelly (mBSK) medium with 12% rabbit serum (65 (link), 66 ). Cultures were centrifuged at 11,000 × g for 20 min at 10°C. Cells were washed twice with phosphate-buffered saline (PBS) plus 5mM MgCl₂ and centrifuged following each wash. Cells were resuspended in a 1:1 solution of PBS plus 5 mM MgCl₂:2× Laemmli sample buffer (SB) (Bio-Rad, Hercules, CA, USA) with 2-mercaptoethanol (BME) (Sigma, St. Louis, MO, USA) at a density of 2 × 10⁶ cells μL⁻¹.