Abstract:

Western blot is the most important method on studying and analyzing protein. Because it contains many steps, high background, unspecific bands, streaking and so on are the common problems in Western blot, which will influence the subsequent detection and quantitation of protein. To establish the best experiment condition for detection of protein expression in rabbit artery, VCAM-1(vascular cell adhension molecule-1) was detected. A rapid, specific and sensitive procedure has been successfully established, through optimizing several conditions which include SDS-PAGE, antibody dilution, temperature and time for antibody incubation, detection methods and so on. Also, three kinds of detection methods were used which contain DAB, enhanced chemilluminescence (ECL) with exposure captured on X-ray film or with cooled charged-coupled device (CCD) cameras. The sensitivity of three methods was compared. The results indicated that ECL has sensitivity 2-10 times that of DAB, and CCD cameras have 4 times sensitivity of X-ray and a more precise procedure. So, ECL-CCD cameras is a promising detection method of Western blot.