An efficient method for olive leaves proteins extraction and two-dimensional electrophoresis

Background: Two-Dimensional Electrophoresis (2-DE) is currently the most common technology used for proteome analysis. The greatest challenge to achieve a well resolved 2-DE map is the protein extract preparation. Evergreen and thick leaves are recalcitrant tissues containing extremely high levels of interfering molecules such as oxidative enzymes, phenolic compounds (simple phenols, flavonoids, condensed tannins, lignin), carbohydrates, nucleic acids and lipids which make the 2-DE analysis problematic. Objective: In the present study, we developed an easy-to-use protocol for optimal olive leaf proteins extraction in high phenolic content condition. Methods: To establish an effective protein extraction protocol, suitable for successive 2-DE processing, the three following protein extraction methods were tested: Phenol, TCA/Acetone-Phenol/SDS and TCA/Acetone-Methanol-Phenol/SDS. Results: Protein spots were undetectable in the 2-DE gel image using phenol extraction method. TCA/Acetone-Methanol-Phenol/SDS protocol showed a higher protein yield, a greater spot number, better spot resolution, higher spot intensities in the high and low molecular weight and minimal streaking on 2-DE gel than the classical TCA/Acetone-Phenol/SDS method. Conclusion: TCA/Acetone-Methanol-Phenol/SDS protocol proved to be the most suitable method to extract proteins from a green recalcitrant phenolic rich tissue as olive leaf (Olea europea L.). Our implemented protocol is expected to be applicable to other recalcitrant plant tissues.