Western Blot Analysis of Sporulating B. subtilis

Whole-cell lysates from sporulating cells were prepared as previously described (Doan and Rudner, 2007 (link)). Samples were heated for 5 min at 65°C prior to loading. Equivalent loading of proteins was based on the OD₆₀₀ of the cell cultures at the time of harvest. Samples were separated on a 12.5% SDS-polyacrylamide gel and transferred to a methanol-activated PVDF membrane. Membranes were blocked in 5% nonfat milk with 0.5% Tween-20 for 1 hour. Blocked membranes were probed with anti-σ^H (diluted 1:2,500), anti-Spo0A (diluted 1:5,000) or anti-SpoIIQ (diluted 1:10,000) (Doan et al., 2009 (link)), anti-σ^A (diluted 1:10,000), anti-KinA (diluted 1:5,000), and anti-core RNA-polymerase (diluted 1:5,000). These primary antibodies were diluted into PBS with 0.05% Tween-20 and incubations were carried out at 4°C overnight. Primary antibodies were detected with horseradish-peroxidase conjugated anti-mouse or anti-rabbit antibodies and detected with Western Lightning ECL reagent as described by the manufacturer (PerkinElmer, Waltham, MA, USA).