Abstract
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 .
Highlights
IntroductionThe extraction and solubilization of proteins for proteomic analysis is a milestone for subsequent analysis by polyacrylamide two-dimension electrophoresis (2D-PAGE) [1]
The results suggest that thiourea/urea and trichloroacetic acid (TCA) methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris
The extraction and solubilization of proteins for proteomic analysis is a milestone for subsequent analysis by polyacrylamide two-dimension electrophoresis (2D-PAGE) [1]
Summary
The extraction and solubilization of proteins for proteomic analysis is a milestone for subsequent analysis by polyacrylamide two-dimension electrophoresis (2D-PAGE) [1]. By optimizing the methods of sample preparation, or the extraction of proteins resolubi-. Lized in a buffer compatible with the electrophoresis, we improved the conditions so that separation by isoelectric focusing (IEF) is possible [1] [2]. Much progress has been made [3] [4] in sample application with the use of immobilized pH gradient strips (IPG) for isoelectric focusing (IEF). The extraction and solubilization are difficult to implement in legumes. The study of legume (soy) proteomics has already been reported [5]; these authors have underlined the difficulty of extracting and solubilizing its proteins
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