The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this ...The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this study, we used two methods of extraction and solubilization of proteins, the urea/thiourea method and the trichloroacetic acid (TCA)/acetone precipitation method, in order to determine their effectiveness in separating proteins from bean seeds by 2D-PAGE. In both methods, proteins were well separated by 2D PAGE with minor variations in the protein pattern. These two extraction methods showed that it was possible to separate hundreds of very resolvent proteins by 2D electrophoresis. A protein spot was selected on the 2D-PAGE gel, digested with trypsin and analyzed by mass spectrometry (LCMS/ MS). The results suggest that thiourea/urea and TCA methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris .展开更多
文摘The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this study, we used two methods of extraction and solubilization of proteins, the urea/thiourea method and the trichloroacetic acid (TCA)/acetone precipitation method, in order to determine their effectiveness in separating proteins from bean seeds by 2D-PAGE. In both methods, proteins were well separated by 2D PAGE with minor variations in the protein pattern. These two extraction methods showed that it was possible to separate hundreds of very resolvent proteins by 2D electrophoresis. A protein spot was selected on the 2D-PAGE gel, digested with trypsin and analyzed by mass spectrometry (LCMS/ MS). The results suggest that thiourea/urea and TCA methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris .
