Horseradish peroxidase conjugated goat anti rabbit igg

S. pneumoniae cultures were normalized to an OD₆₀₀ of 0.3 and 3 ml harvested. Cell extracts were prepared by resuspension of cell pellets in 100 μl lysis buffer (20 mM Tris pH 7.5, 10 mM EDTA, 1 mg ml⁻¹ lysozyme, 10 μg ml⁻¹ DNase I, 100 μg ml⁻¹ RNase A, with protease inhibitors: 1 mM PMSF, 1 μg ml⁻¹ leupeptin, 1 μg ml⁻¹ pepstatin) and incubation at 37°C for 10 min, followed by addition of 10 μl 10% Sarcosyl for 5 min. 100 μl SDS sample buffer (0.25 M Tris pH 6.8, 4% SDS, 20% glycerol, 10 mM EDTA) containing 10% 2-mercaptoethanol was added to each prep and samples were heated for 15 min at 50°C prior to loading 10 μl per lane. Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto a PVDF membrane and blocked in 5% non-fat milk in PBS-0.5% Tween-20. The blocked membranes were probed with rabbit anti-FtsE (1:20,000) ^{33 (link)} and affinity-purified rabbit anti-GFP (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 Lighning Plus ECL reagent as described by the manufacturer (PerkinElmer). Membrane chemiluminescence was imaged on a FluorChem R system (ProteinSimple).