Semi dry transfer blot apparatus

Protein samples were subjected to SDS–PAGE (10% [w/v] acrylamide) according to the method of Laemmli (Laemmli, 1970 (link)) at room temperature for 2 h at 100 V in a Mini-Protean ^®III-2D cell (Bio-Rad). After electrophoresis, proteins on the gels were stained with Coomassie Blue R-250 or electroblotted onto a nitrocellulose membrane (N-8017, Sigma) at 10 V (5.5 mA cm^-2) for 30 min in a semidry transfer blot apparatus (Bio-Rad Laboratories). Membranes were blocked in Tris-buffered saline (20 mM Tris–HCl and 0.15 mM NaCl [pH 7.5]) containing 5% (w/v) powdered milk, and bands were immunochemically labeled by overnight incubation of the membrane at 4°C in 20 ml of Tris-buffered saline containing specific antibodies. Subsequent detection was performed using a horseradish peroxidase conjugated antibody (Bio-Rad) by a peroxidase assay (Figure 1, 2, 3, 5) or by a chemiluminescence detection system (Super Signal West Dura Signal; ThermoFisher, Waltham, MA, United States) according to the manufacturer’s instructions (Figure 4).

An overnight culture was diluted in LB to OD 0.25 and grown for three hours with and without inducers (107 nM aTc and 1 mM IPTG). The sample with inducers was split and diluted again (to OD 0.25) to create two samples, one with 107 nM aTc and 1 mM IPTG and the other with 107 nM aTc, 1 mM IPTG and 100 nM AHL. Before and after the dilution, 2 ml from each sample were pelleted and suspended in lysis buffer (50 mM Tris, 14 mM MgGlu, 60 mM KGlu, 1 mM DTT, 0.1% TritonX100, pH 7.7) so that each sample had a concentration of 5 μg/ml (calculated from the OD). The samples were lysed with sonication on ice, pelleted and the supernatant was denatured at 95°C in Lämmli buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 20 μg of proteins were transferred to PVDF membranes using a semi-dry transfer-blot apparatus (Bio-Rad). The membranes were blocked with 5% (w/v) BSA in TBST overnight at 4°C and probed with anti-sera to FtsZ (1:1000, Agrisera) for 1,5 h at room temperature [47 (link)]. A TRITC anti-mouse secondary antibody (1:5000; Agrisera) was applied for 1 h at 4°C in 5% BSA in TBST for 1 h and the blot was imaged with a Typhoon FLA 9500 scanner (General Electric) in the Cy3 channel.