Abstract: Objective The aim of this study is to compare protein expression profiles in two-dimensional electrophoresis analysis using different protein extraction protocols.Methods Total proteins were extracted from Hela cells with freezing-thaw,ultrasonic treatment,gentle shake treatment and 2-D clean up kit respectively.Samples were separated by two-dimensional electrophoresis,in which 300 μg of protein samples were applied to 7cm pH 3-10 NL immobilized pH gradient gel strips.Protein spots were visualized by modified colloidal CCB staining and then analyzed with Image Master 2D Platinum Software.Results Samples from freezing-thaw treatment exhibit fewer protein spots(400±) and more horizontal stripes on 2-DE maps.In contrast,more spots(700± and 800±) and fewer horizontal stripes were detected with ultrasonic or gentle shake treatment,and stripes were further reduced with 2-D clean up kit.However,2-D clean up kit detected less protein spots especially for low-abundance proteins,and addition of Destreak reagent to 2-D clean up kit can increase the efficiency of protein yield on the 2-D image(800±→900±).Conclusion Protein expression profiles of Hela cells in 2-DE analysis were obtained with few stripes and more protein spots after ultrasonic or gentle shake treatment to extract proteins.Combination of 2-D clean up kit with Destreak reagent for protein extraction can diminish horizontal stripes,especially in the alkaline end.

Key words: two-dimensional electrophoresis, protein expression profile, protein extraction