Programa de Pós-Graduação em Ciências Biológicas - Centro de Ciências Biológicas - Universidade Federal de Pernambuco, Rua Prof. Nelson Chaves s/n, Cidade Universitária, CEP 50670-901 Recife, PE Brasil.
Programa de Pós-Graduação em Fitopatologia - Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros, s/n - Dois Irmãos, CEP: 52171-900 Recife, PE Brasil.
Proteome Sci. 2015 Feb 5;13:5. doi: 10.1186/s12953-015-0062-9. eCollection 2015.
Finding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels.
The bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli, the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels.
These results attest to the adequacy of these proteins extraction methods for proteomic studies.
在所有蛋白质组学应用中，从裂解细胞中找到最佳的蛋白质提取方法是检测和鉴定的关键步骤。这些方法对于补充有关导致重大经济损失的植物病原体之间相互作用机制的知识非常重要。为了开发优化的提取方案，使用酸噬细胞菌、果胶杆菌亚种胡萝卜软腐病菌和茄青枯雷尔氏菌作为植物病原菌的代表细胞进行研究。本研究旨在比较四种不同的蛋白质提取方法，包括 Trizol、酚、离心和裂解，以确定哪种方法更适合使用二维凝胶观察到的提取蛋白质的数量和质量作为参数的蛋白质组学研究。
所研究的细菌在测试方法之间表现出不同的结果。裂解法对果胶杆菌亚种胡萝卜软腐病菌和茄青枯雷尔氏菌更有效，既简单又快速，而对于酸噬细胞菌，离心法是最佳的。这种评估是基于聚丙烯酰胺凝胶中获得的结果，二维凝胶检测到的斑点数量更多，条带更清晰、更一致。
这些结果证明了这些蛋白质提取方法适用于蛋白质组学研究。
