Immunoblotting Techniques for Bacterial Proteins

Equivalent OD units of cell lysate were loaded on an SDS-PAGE gel following cell harvest by centrifugation, lysing 1X SDS loading dye, and boiling for 5 – 10 minutes. 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 (Regis 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).