1College of Agronomy and Biotechnology,Southwest University,Plant Physiology and Biochemistry Laboratory,Southwest University,Chongqing 400716,China|2College of Horticulture and Landscape Architecture,Southwest University,Key Laboratory of Horticulture Science for Southern Mountainous Regions,Ministry of Education,Chongqing 400715,China
Abstract:A two-dimensional electrophoresis(2-DE)has been widely applied in proteomics research.The purpose of this study was to establish a suitable 2-DE system for proteomic analysis of Brassica oleracea L. var. capitata L. stigma.The stigmas being harvested one day before flowering was used in the experiment.We optimized the parameters of 2-DE including extraction methods,IPG strip,sample loading,isoelectric focusing conditions and SDS-PAGE gel concentration.The optimized system included the following steps:extracting the total proteins from Brassica oleracea stigma by TCA/acetone precipitation,separating the proteins with length 17 cm and pH 3-10 NL IPG strips,loading proteins samples of 150 μg followed by isoelectric focusing programⅠ,and selecting SDS-PAGE gel concentration at 12%.The 2-D images with good repeatability and high protein spot resolution were obtained.