Antisera to ftsz

Cell extracts from a volume corresponding to 0.2 OD₆₀₀ units were collected for different strains. The extracts were suspended in loading buffer and resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes using a semi-dry Turbo Transfer-Blot apparatus (Bio-Rad). The membranes were blocked in 5% (w/v) milk and probed with antisera to FtsZ [1:4000] (Agrisera, Sweden).

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.