Establishment And Application Of Two-dimensional Electrophoresis Technique For Human Colorectal Carcinoma Tissues Proteome Profiling

Objective The application of two-dimensional electrophoresis (2-DE) technique for proteome profiling to human colorectal carcinoma tissues is more important than to colorectal carcinoma cell lines or animal models. There is few reports abroad and no report in China on the application of 2-DE technique to human colorectal carcinoma tissues proteome profiling . The thesis aims to establish a 2-DE technique through which 2-DE patterns with high resolving power and reprbducibility can be obtained for the tissues proteome profiling.Methods In sample preparation, three different volume rates of tissue to lysis liquid were compared, two kinds of lysis liquid (with or without thiourea and TritonX-100) and three kinds of sample protein weight (200ug ,400ug,600ug) were compared too. In the first dimension, immobilized pH gradient(IPG) gel strip was used and three kinds of time-voltage sequence for isoelectric forcusing were compared. In the second dimension, SDS-PAGE was used. After comparation, an optimized technique was established and then carried out three times. ImageMaster 2D Elite analysis software was utilized to evaluate the image quality in resolving power and reproducibility.Results In sample preparation, it was adequate as volume rate of tissue to lysis liquid to be 1 to 8. When thiourea and TritonX-100 were added to the lysis liquid, more protein spots wre obtained.The map was best when sample protein weight is 400ug. The most adequate time-voltage sequence of isoelectric focusing was 30V16hr, 500Vlhr, 1000Vlhr, 3500Vlhr, 8000V6h, total 53480Vhr. Using ImageMaster 2D Elite analysis software , three different images carried by optimized technique were analysized. The average number of total protein spots was 1086?4, the average deviations for protein position in IEF direction is 1.98+0.39mm, SDS-PAGE direction 1.81+0.26mm, the relative standarddeviations for protein volume is 7.67%+2.75%.Conclusion Using IPG gel strip and with adequate time-voltage sequence in the first dimension, SDS-PAGE in the second dimension, optimizing volume rates of tissue to lysis liquid, adding thiourea and TritonX-100 to lysis liquid, sample protein weight being 400ug , the 2-DE technique can obtain images with high quality in resolving power and reproducibility for colorectal carcinoma tissue proteomics research.