Immunoblotting Analysis of LptD and σE

Rabbit polyclonal antibodies against S. oneidensis LptD, which were prepared using a synthesized fragment (amino acids [aa] 40 to 198) as the antigen in accordance with standard protocols provided by the manufacturer (GenScript, Nanjing, China), and antibodies against σ^E prepared previously (14 (link)) were used for immunoblotting analysis. Sample preparations, including cell cultivation and subcellular fractionation, were carried out as described before (14 (link), 63 (link)). Throughout this study, the total protein concentration of the cell lysates was determined by the bicinchoninic acid assay (Pierce Chemical). The resulting samples for defection of LptD and σ^E were subjected to electrophoresis on 6% and 15% SDS polyacrylamide gels (PAGE), respectively. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes for 1 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with specific antibodies, followed by a 1:10,000 dilution of goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate. The alkaline phosphatase was detected using a chemiluminescence Western blotting kit (Roche Diagnostics) in accordance with the manufacturer’s instructions. Images were visualized with Clinx Imaging System (Clinx, Shanghai, China).