Immun star horseradish peroxidase hrp substrate kit

The supernatant after pelleting dislodged EC and ECDPS was used to determine the endothelial marker of VWF using commercial antibodies. HF sheep plasma samples are also analyzed. Protein concentration was determined by using Bio-Rad DC protein assay (Hercules, CA). Protein of 8-10 mg was separated on 8% SDS-PAGE. The gels were transblotted onto a 0.2-mm nitrocellulose membrane using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) for 1 h at 100V in a cold room. Membranes were blocked with 5% nonfat milk in Tris-buffered saline (pH 7.5) with 0.1% Tween-20 at room temperature for 1 h and then incubated with primary antibody to VWF (rabbit raised polyclonal antibody; 1:5000 v/v dilution) overnight at 4 C. The blots were then incubated with secondary antibody (goat raised polyclonal to rabbit IgG; 1:5000 v/v dilution) for 1 h. Finally, signals were detected with Bio Rad Immun-star horseradish peroxidase (HRP) substrate kit (Hercules, CA). Densitometric analysis was performed by using UN-Scan IT-gel 6.1 version.