Secondary hrp labeled anti rabbit antibody

Western blot analysis of LtrB relaxase was performed as previously described (17 (link)). Briefly, total cell lysate was separated on a 12% SDS-polyacrylamide gel and then transferred to 0.2 μM immuno-blot PVDF membrane (Bio-Rad). The membrane was blocked with 5% milk in TBS-T (20 mM Tris–HCl (pH 7.5), 140 mM NaCl, 2% Tween 20), incubated for 1 h with a 1/2,000 dilution of primary anti-relaxase antibody (gift from Gary Dunny, University of Minnesota), washed with TBS-T and incubated for 1 h with a 1/10 000 dilution of secondary HRP-labeled anti-rabbit antibody (Advansta). The imaging was done using chemiluminescent HRP substrate (Advansta WesternBright ECL) under a Bio-Rad ChemiDoc MP. For normalization, total protein from a Coomassie-stained 12% SDS-polyacrylamide gel was used as loading reference. Quantitation was done using Image Lab (Bio-Rad) software. For analysis of the IEP, after the 3-h induction, the same procedures were followed except that an anti-IEP antibody (Covance) was used as the primary antibody.