Protein Purification and Western Blotting

Total proteins were purified in the absence of detergent [43 (link)]. Briefly, samples containing spores were pelleted by centrifugation and rinsed with 1× PBS. Total protein concentration was measured using the bicinchoninic acid assay (BCA) using protocols from the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were electrophoretically separated in 10% polyacrylamide-SDS gels using a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad^®, Hercules, CA, USA) [40 (link)]. The proteins were transferred to 0.45 μm nitrocellulose (Bio-Rad^®, Hercules, CA, USA) with a Trans-Blot Semi-Dry Transfer system (Bio-Rad^®, Hercules, CA, USA). Table 1 shows the tested antibodies. Following transfer, immunoblots were viewed with enhanced chemiluminescence (ECL) substrates (Bio-Rad^®, Hercules, CA, USA) using a Vilber Lourmat gel doc Fusion FX5-XT (Vilber^®, Marne-la-Vallee, France). Densitometry was conducted with FUSION FX software (Vilber^®, Marne-la-Vallee, France), using total protein to normalize measurements. Data represent three separate experiments from three different total protein isolations.