S. pneumoniae strain DSM20566 (serotype 1) was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). Bacteria were grown on Columbia blood agar plates (BD Biosciences, Heidelberg, Germany) at 37°C in 5% CO2 overnight or cultured in brain heart infusion (BHI) broth (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) at 37°C as described elsewhere (Walther et al., 2015 (link)).
Precipitation of pneumococcal total proteins was described previously (Richter et al., 2015 (link)). Briefly, 200 μL of a pneumococci culture (grown overnight in BHI broth) were precipitated by 1.8 mL of ice-cold absolute ethanol (−20°C for 16 h). Aliquots were centrifuged at 3000 × g for 20 min at 4°C. The precipitate was washed with 1 mL of ice-cold 70% ethanol. After centrifugation at 3000 × g for 5 min at 4°C, the supernatant was removed and the pellet dried. The ethanol-precipitated proteins were dissolved in 50 μL of NA assay buffer and stored at −20°C until use.
Precipitation of pneumococcal total proteins was described previously (Richter et al., 2015 (link)). Briefly, 200 μL of a pneumococci culture (grown overnight in BHI broth) were precipitated by 1.8 mL of ice-cold absolute ethanol (−20°C for 16 h). Aliquots were centrifuged at 3000 × g for 20 min at 4°C. The precipitate was washed with 1 mL of ice-cold 70% ethanol. After centrifugation at 3000 × g for 5 min at 4°C, the supernatant was removed and the pellet dried. The ethanol-precipitated proteins were dissolved in 50 μL of NA assay buffer and stored at −20°C until use.
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