An efficient 2-D gel electrophoresis protocol suitable for seed proteome of rice

Abstract

The aim of this article was to provide the efficient protocol for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) suitable for the seed proteome of rice. Sample preparation is a crucial step in 2D-PAGE because it directly affects the quality of protein profiles. We investigated the two different methods to extract the proteins viz., direct lysis buffer method and trichloroacetic acid/acetone (TCA/acetone) method. The effectiveness was evaluated by the protein profing quality, number of protein spots, consistency and distribution of protein spots over the gels. The protein extracted by TCA/acetone method had the highest number of protein spots and the good resolution of the gels. Two different IEF focusing were tested in which, the protein run at 50000 Volt hours showed the clear protein pattern and more number of proteins than 35000 Volt hours. Compared to the Coomassie brilliant blue (CBB) G-250 staining, silver nitrate staining produced the more number of proteins spots. The protocol using TCA/Acetone extraction with 50000 Volt-hour IEF conditions followed by the silver staining resulted in the best extraction efficiency, greater protein separation and less horizontal streaking in 2DE gels. This works could principally provide the useful information and technical support for the rice seed proteomic research.