Cobalt Affinity Purification (Pull-Down Assays).

E. coli cotransformed with two plasmids were grown in 500 mL of LB medium and induced as described above. Cells were harvested, lysed, and centrifuged at low speed as described (26) except 20 mL of lysis buffer was used. The supernatant was treated with 1% n-decyl-β-d-maltoside (DM) (Anatrace) for 1 h at 4 °C to solubilize membrane proteins then centrifuged at 150,000 × g for 1 h at 12 °C. The supernatant was designated the input sample, and 100 μL was saved for immunoblot analysis. The rest was mixed with 0.5 mL of Talon superflow metal affinity resin (Clontech) that had been equilibrated with PBS containing 0.1% DM, 5 mM 2-mercaptoethanol, and 10% glycerol. The mixture was rotated for 1 h at room temperature. The cobalt resin was sedimented by centrifugation at 708 × g for 2 min at 4 °C. The supernatant was the unbound sample. The resin was washed three times with 5 mL of PBS containing 150 mM NaCl and 10% glycerol, then once with 5 mL of PBS containing 150 mM NaCl, 10% glycerol, 0.1% DM, and 40 mM imidazole, each time rotating the mixture briefly and sedimenting the resin as above. The resin was mixed with 10 mL of PBS containing 150 mM NaCl, 10% glycerol, and 0.1% DM, resulting in the bound sample. The samples (75 μL) were added to 25 μL of 4× sample buffer (100 mM Tris·HCl pH 6.8, 8% SDS, 40% glycerol, 400 mM DTT, and 0.06% bromophenol blue), boiled 3 min, and subjected to immunoblot analysis as described above. A representative result from at least two biological replicates is shown. Signal intensities were quantified using Image Lab Software (Bio-Rad) for two replicates, and the range of ratios of bound/unbound SpoIVFB is reported in Fig. 2F.