Horseradish peroxidase hrp conjugated anti mouse igg

Crude GBS whole cell (WC) extracts, purified rSip or nature Sip were subjected to 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots. For WC samples, a 10 ml overnight culture was centrifuged to obtain the pellet, which was added with 25 μl of sample buffer (0.2 M Tris-HCl (pH 6.8), 1% SDS, 2% mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) and then boiled for 5 min, and after centrifugation, 10 μl of supernatant was applied to the gel. After electrophoresis, protein bands were visualized by staining with coomassie brilliant blue (CBB) or transferred to nitrocellulose membranes for western blotting method using anti-rSip McAbs. For western blotting, the membrane was treated as follows: blocked with phosphate-buffered saline (PBS, pH 7.4) containing 3.0% BSA (Sigma-Aldrich, Beijing, China) and 0.1% Tween 20 (Sigma-Aldrich, Beijing, China) for 1 h at 24 °C, washed 3 times with PBS containing 0.1% Tween 20, incubated with 100 ng/ml of anti-rSip antibodies at 4 °C for 1 h, washed 3 times with PBS containing 0.1% Tween 20, incubated with 2,000-fold-diluted horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma-Aldrich, Beijing, China) at 24 °C for 1 h, washed 3 times with PBS containing 0.1% Tween 20, and then soaked in the TMB membrane peroxidase substrate system (Sigma-Aldrich, Beijing, China) for color development.