Western Blot Protein Detection Protocol

Immunoblotting samples were prepared by harvesting 0.5 or 1 mL cells by centrifugation and resuspending pellets in OD₆₀₀/0.003 or OD₆₀₀/0.003 μL 1× sodium dodecyl sulfate (SDS) loading dye, respectively. Equivalent OD units of cell lysate were loaded on an SDS–-polyacrylamide gel electrophoresis (PAGE) gel following cell harvest by centrifugation, lysing 1× SDS loading dye, and boiling for 5–10 min. SDS–PAGE and protein transfer to nitrocellulose membranes were followed using standard procedures. Antibodies were used at the following dilutions: CdnL 1:10,000 (14 (link)); MreB 1:10,000 (Régis Hallez, University of Namur); SpmX 1:20,000 (Patrick Viollier, University of Geneva); GFP 1:2,000 (Clonetech Labs, catalog #NC9777966); HRP-labeled α-rabbit secondary 1:10,000 (BioRAD, catalog #170-6515); and/or HRP-labeled α-mouse secondary (Cell Signaling Technology, catalog #7076S). Clarity western electrochemiluminescent substrate (BioRAD, catalog #170-5060) was used to visualize proteins on an Amersham Imager 600 RGB gel and membrane imager (GE).