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A novel method of protein extraction from perennial Bupleurum root for 2‐DE

The perennial Bupleurum root is thick and woody and contains high levels of interfering compounds. Common protein extraction methods have proved refractory towards the isolation of proteins suitable for 2‐DE, due to the presence of interfering compounds. A novel method for extracting proteins suitable for 2‐DE was established to overcome these problems. The main characteristic of this protocol is the partitioning of the proteins into the aqueous (fraction A‐2), chloroform and isoamyl alcohol phases (A‐3), and the interphase (A‐1). The proteins are then extracted from each of these phases. From A‐1, 85% (extracted protein against total proteins) proteins could be extracted and purified. For fraction A‐2, a novel phenol extraction step is employed for the extraction of proteins. Based on the well‐resolved 2‐DE patterns, our protein preparation is free of interfering compounds. Using these methods (A‐1, A‐2, and A‐3‐3), a total of 3662 (1526 + 1128 + 1008) spots could be separated, and a protein yield of about 1.41 mg per 1.0 g fresh root material was obtained. To our knowledge, this is the first time that a protocol for protein extraction from perennial Bupleurum root has been reported that gives reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues as well.