An Analysis Of Western Blotting For Detection Of B7 Protein In Liver Of Patient With Hepatic Cirrhosis

Objective: In order to establish the method for detection B7.1 or B7.2 protein in liver of patient with hepatic cirrhosis, Western blotting anlysis was explored.Methods: Western blotting analysis was used in the patients with hepatic cirrhosis. In order to determinate the optimum dilution of the primary antibody for B7.1 or B7.2, gradient dilution of antibody, namely 1:1000,1:500,1:200,1:100 and 1:50, were operated. To establishing optimum exposing time of the X-ray film, gradient exposing time, namely 7.5, 15, 30, 60 and 120minute, also were applied. Positive control, negative control, prestained protein marker and recombiant protein were co-electrophoresised in the same PVDF membrane. The molecular mass and specificity of B7.1 or B7.2 were determinated.Results:By the controlling of positive and negative controls, prestained protein marker and recombiant protein, B7.2 protein was expressed in liver of patient, optimum condition were 1:200 B7.2 antibody dilution and 60 minute X-ray film exposing time. And also, the optimum condition for B7.1 protein was 1:50 B7.1 antibody dilution and 120 minute X-ray film exposing time. The molecular mass of B7.2 is 75KD, and B7.1, 60KD.Conclusion: The method of Western blotting for detecting in liver of patient is higher specific and sensitive, it is useful for the studies of the function of B7.