Phenol extraction of proteins for proteomic studies of recalcitrant plant tissues

Phenol extraction of proteins is an alternative method to classical TCA-acetone extraction. It allows efficient protein recovery and removes nonprotein components in the case of plant tissues rich in polysaccharides, lipids, and phenolic compounds. We present here a tried and tested protocol adapted for two dimensional electrophoresis (2-DE) and further proteomic studies. After phenol extraction, proteins are precipitated with ammonium acetate in methanol. The pelleted proteins are then resuspended in isoelectric focusing buffer, and the protein concentration is measured with a modified Bradford assay prior to electrophoresis. The important points for successful use of this protocol are (1) keeping samples at very low temperature during the first step and (2) careful recovery of the phenolic phase after the centrifugations, which are major features of this protocol.