Western Blot Analysis of Sporulating Cells

Whole-cell lysates from sporulating cells (induced by resuspension) were prepared as described previously (Doan et al., 2009 (link)). Samples were heated for 10 min at 50°C prior to loading. Equivalent loading was based on OD₆₀₀ at the time of harvest. Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto Immobilon-P membranes (Millipore) and blocked in 5% nonfat milk in phosphate-buffered saline (PBS)-0.5% Tween-20. The blocked membranes were probed with anti-SpoIID (1:10,000) (Doan & Rudner, 2007 (link)), anti-SpoIIQ (1:10,000) (Doan et al., 2005 (link)), anti-SpoIIIAH (1:10,000) (Doan et al., 2005 (link)), anti-SpoIIP (1:10,000) (Morlot et al., 2010 (link)), anti-σA (1:10,000) (Fujita, 2000 (link)), anti-EzrA (1:10,000) (Levin et al., 1999 (link)), anti-His (Genscript) (1:4000), anti-mCherry (1:10,000), diluted into 3% BSA in 1X PBS-0.05% Tween-20. Primary antibodies were detected using horseradish peroxidase-conjugated goat, anti-rabbit IgG (1:20,000, BioRad) and the Western Lightning reagent kit as described by the manufacturer (PerkinElmer).