Capsule Detection via Blotting Assay

Capsule presence was detected using the capsule blotting method [27 (link)]. Briefly, strains were grown to mid-logarithmic phase (OD₆₂₀ = 0.4) in C+Y and were lysed using DOC/SDS disruption of the membrane. Proteins were removed by incubation with proteinase K; a small sample of the cell lysate was used to measure protein concentration prior to protein degradation via BCA assay (Pierce). Cell lysates were diluted to 22.5 mg/mL cellular protein and subjected to agarose gel electrophoresis. The samples were transferred to a nitrocellulose membrane following standard capillary transfer in a high-salt buffer. The membrane was then probed with serotype-specific antibodies (Statens Serum Institute) and HRP conjugated secondary antibody (Bio-Rad), followed by detection using a HRP chemiluminescent substrate (Thermo Scientific).