Amersham imager 600 detection system

To validate the accuracy of iTRAQ results, western blot analysis was performed.
The plasma was diluted ten times by PBS. 30 μL of samples per lane were loaded for 10% Bis-Tris polyacrylamide gels and then transferred onto PVDF membrane (Millipore, Billerica, MA) by electroblotting. The membranes were blocked in TBS 0.5% Tween containing 5% skim milk powder (OXOID, Basingstoke, UK) for 1 h at room temperature and incubated with primary antibody: anti-IGFBP4 (Abcam, Cambridge, UK) overnight at 4 °C. Then the membranes were incubated with peroxidase-conjugated secondary antibodie (Bio-Rad, Hercules, USA) for 1 h at room temperature. The bands were visualized by using Millipore’s enhanced chemiluminescence (ECL) with the Amersham Imager 600 detection system (GE, Boston, USA). The western blot results represent at least 2 independent biological replicates.

Bacteria (10⁹ cfu) were lysed in 200 μl lysis buffer (from the protein purification kit). Samples of 20 μl were loaded onto 12.5% Bis-Tris polyacrylamide gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA) by electroblotting. The membranes were blocked in Tris-buffered saline and 0.5% Tween containing 5% skimmed milk powder (OXOID, Basingstoke, UK) for 1 h at room temperature and incubated with primary antibody, anti-his (M20001; Abmart, Shanghai, China) with 1:1000 dilution (500 µg/ml) overnight at 4 °C. The membranes were incubated with peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. The bands were visualized using Millipore’s enhanced chemiluminescence with the Amersham Imager 600 detection system (GE, Boston, USA).

Cells were harvested at indicated times and rinsed twice with PBS. The cell extracts were prepared using lysis buffer and centrifuged at 13,000 g for 30 min at 4°C. The protein samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) in 4–20% (wt/vol) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After the membranes were blocked with 5% BSA for 2 h at room temperature, they were incubated with 1.0 μg/mL antibody in 5% BSA overnight at 4°C. The membranes were then incubated with a secondary antibody conjugated to horseradish peroxidase. The signals were detected by electrochemiluminescence reagent. Protein bands were visualized in Amersham Imager 600 detection system (GE Chalfont, United Kingdom).