Polyvinylidenfluoride membranes

Protein samples were separated by SDS–polyacrylamide gel electrophoresis and blotted on polyvinylidenfluoride membranes (Merck Millipore). Membranes were blocked overnight with Tris-buffered saline 1X (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 8) containing, 0.1% Tween-20 and 8% dry milk and then incubated for an additional 3 hr with the primary antibodies diluted in TBS 1X, 0.1% Tween-20, 5% dry milk. The different polyclonal antisera to ZitP (NTER, 1:5000 dilution and CTER, 1:5000), CtrA (1:10,000) and DivJ (1:10,000) were used. Commercial and polyclonal antibodies to Dendra2 (Antibodies-Online) and mCherry (Chen et al., 2005 (link)) were used at 1:5,000 and 1:10,000 dilutions respectively. Primary antibodies were detected using HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) with ECL Western Blotting Detection System (GE Healthcare) and a luminescent image analyzer (Chemidoc^Tm MP, Biorad).

Protein samples were separated by SDS–polyacrylamide gel electrophoresis and blotted on polyvinylidenfluoride membranes (Merck Millipore). Membranes were blocked overnight with Tris-buffered saline 1X (TBS) (50 mM Tris-HCl, 150 mM NaCl [pH 8]) containing, 0.1% Tween-20% and 8% dry milk and then incubated for an additional three hours with the primary antibodies diluted in TBS 1X, 0.1% Tween-20, 5% dry milk. The different polyclonal antisera to CitA (1:5000) and to CtrA (1:5000) were used. Primary antibodies were detected using HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) with ECL Western Blotting Detection System (GE Healthcare) and a luminescent image analyzer (Chemidoc MP, Biorad).