For western blotting after resolving the IpaD protein sample was transferred onto the PVDF membrane (Millipore) using semidry transfer apparatus (Biorad) for 1 hour with constant voltage at 20V. After complete transfer membrane was washed with TBST buffer (20 mM Tris pH7.5, 150mM NaCl and 0.1% (v/v) Tween-20
(Sigma) and then blocked with blocking buffer (3% BSA in TBST buffer) for 1 hour at room temperature with gentle shaking. The membrane was then incubated with mouse anti-IpaD monoclonal antibody (1:1000, Abbexa, USA) in TBST containing 1% BSA overnight at 4 °C. Next, the membrane was washed for three times with TBST then incubated with anti-mouse secondary antibody conjugated with HRP (IgG-HRP) (1:5000; Invitrogen, USA) for 1 hour at room temperature in 3% (w/v) BSA in TBST. After washing thrice with TBST, immunoblot was developed in the presence of a chemiluminescence substrate (Bio-Rad).
(Sigma) and then blocked with blocking buffer (3% BSA in TBST buffer) for 1 hour at room temperature with gentle shaking. The membrane was then incubated with mouse anti-IpaD monoclonal antibody (1:1000, Abbexa, USA) in TBST containing 1% BSA overnight at 4 °C. Next, the membrane was washed for three times with TBST then incubated with anti-mouse secondary antibody conjugated with HRP (IgG-HRP) (1:5000; Invitrogen, USA) for 1 hour at room temperature in 3% (w/v) BSA in TBST. After washing thrice with TBST, immunoblot was developed in the presence of a chemiluminescence substrate (Bio-Rad).