Western Blot Analysis of TonB1 and DdvT

A peptide corresponding to residues 199–210 (QAGNPIRTKDRR) of TonB1 was synthesized and used as an antigen to obtain antisera for TonB1 in rabbits (Cosmo Bio, Inc.). Anti-TonB1-peptide antibodies were obtained by purifying the antiserum using peptide affinity column chromatography (Cosmo Bio, Inc.). Anti-DdvT antibodies were obtained in a previous study^{24 (link)}. Total membrane fractions were prepared as described previously from SYK-6 cells incubated in LB for 20 h^{24 (link)}. TonB1 and DdvT were detected by western blot analysis using anti-TonB1 antibodies (0.11 µg ml⁻¹) and anti-DdvT antibodies (0.25 µg ml⁻¹) as described previously^{24 (link)}. Horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (Invitrogen, 0.2 µg ml⁻¹) were used as the secondary antibodies. Protein concentrations were determined by the Bradford method using a Bio-Rad protein assay kit or Lowry’s assay with a DC protein assay kit (Bio-Rad Laboratories). TonB1 and DdvT were detected using the ECL Western Blotting Detection System (GE Healthcare) with a LumiVision PRO image analyzer (Aisin Seiki Co., Ltd).