Western Blot Analysis of Plasma Proteins

The plasma protein (20 μg) from CAD and healthy controls was separated on 12% SDS-PAGE as described previously [25 (link)] and electrotransferred to the nitrocellulose (NC) membrane (Millipore, USA) using Trans-Blot Semi-dry transfer unit (Bio-Rad, USA). The membranes were blocked using 5% of bovine serum albumin (BSA) (Sigma-Aldrich, USA) for 1 h at RT. Thereafter, NC membranes were incubated overnight at 4°C with their respective primary antibodies: serotransferrin, talin-1, α-2HS glycoprotein, TTR ,and fibrinogen-α chain with 1 : 4000 dilution and with horseradish peroxidase- (HRP-) conjugated anti-mouse secondary antibody (Jackson, USA), at RT for 1 h with 1 : 8000 dilutions on the gentle shaking condition. Each membrane was then developed with enhanced chemiluminescence (ECL) (Thermo Scientific, Pierce, USA), a highly sensitive substrate detector using the Chemi-Doc_TM MP Imaging system (Bio-Rad, USA). After each incubation, membranes were washed thrice with wash buffer (0.05% tween-20 in a phosphate buffer saline).