Hrp conjugated immunoglobulin

The expression of Ang-2 was measured in pancreatic cancer cell lines (BxPC-3, MiaPaCa-2 and Panc-1) or tumor tissue by western blotting. Protein extraction was performed using the extraction buffer containing 10 mM Tris HCl (pH 7.5), 2 M urea, 2 mM EDTA, 2 mM EGTA, and protease inhibitors. Thirty micrograms of protein were loaded onto SDS–polyacrylamide gels, size fractionated on SDS-PAGE gels, and transferred to a nitrocellulose membrane using the semidry technique. The membranes were blocked with 5 % milk powder in TBST for 1 h. Specific monoclonal anti-Ang-2 (ab8452) and monoclonal anti-β-actin (ab3280) primary antibodies (Abcam Biotechnology, Cambridge, MA, USA) were used, and HRP conjugated immunoglobulin was used as a secondary antibody (Jackson ImmunoResearch Laboratories). West Pico Chemiluminescent (Pierce) was used as the substrate to visualize protein bands, which were quantified using densitometry image analysis software (Image Master VDS; Pharmacia Biotech). Normalization was made against β-actin expression.

Thirty microgram of cell lysates and tumor tissue lysates were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto nitrocellulose membranes. Specific monoclonal anti-UCP-2 (ab67241) and monoclonal anti-β-actin (ab3280) primary antibodies (Abcam Biotechnology) were used, and HRP conjugated immunoglobulin was used as a secondary antibody (Jackson ImmunoResearch Laboratories). West Pico Chemiluminescent (Pierce) was used as the substrate to visualize protein bands, which were quantified using densitometry image analysis software (Image Master VDS; Pharmacia Biotech).