P38 PROTEIN EXTRACTION OF POPPY (PAPAVER SOMNIFERUM) FOR PROTEOMIC 2 – DE ANALYSIS Tímea Kuťka Hlozáková 1 , Edita Gregová, Svetlana Šliková 2 , Zdenka Gálová 1 1 Slovak University of Agriculture, Faculty of Biotechnology and Food Science, Nitra, Slovakia 2 National Agriculture and Food Centre, Research Institute of Plant Production, Piešťany, Slovakia xhlozakova@is.uniag.sk 1 Introduction Strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. Three different extraction procedures for two-dimensional electrophoresis of poppy are compared in this work. TCA/acetone-based and phenol- based extraction method, have been mainly used to extract proteins from different organs or tissues on many plant species. However, few results have been reported for poppy or another oilseed plants. We wanted to determine which of these protocols was optimal for oilseed plants in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. The phenol-based protocol was superior to the TCA/acetone and sodium phosphatase methods, showing larger protein yields and greater spot resolution on 2-D gels. 2 Experimental 2.1 Sample Seeds were used for extraction of proteins from seven Slovak registrated (Albín, Belgarn, Gerlach, Manor, Malsar, Maratón, Opál) and one Hungarian registrated (Buddha) varieties of poppy, which were obtained from GeneBank of Slovak Republic in Piešťany. 2.2 Extraction protocols The first protocol used for extraction of the proteins was using phenol followed methanolic ammonium acetate precipitation [1– modified]. Plant tissue (100 mg) was homogenized well in the extraction buffer (0.1 M Tris – HCl pH 8.8, 10 mM EDTA, 0.4 % 2 – mercaptoethanol, 0.9 M sucrose) and the same volume of 0.4 M phenol buffer pH 8.8 was added. This mixture was shaken vigorously for 30 min at 4ºC and then centrifuged at 5000g for 10 min at 4ºC. The upper phenol phase containing the proteins was collected very carefully. Ammonium acetate (0.1 M) was added five times the volume of the phenol phase. Mixed well and kept for precipitation overnight at – 20ºC. Next day, the mixture was centrifuged at 5000 g for 20 min at 4ºC. The supernatant was discarded and precipitates were washed in 0.8 M acetone twice and once in 0.7 M ethanol. 228 DEMO : Purchase from www.A-PDF.com to remove the watermark