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Protein extraction is a critical step for two-dimensional electrophoresis(2-DE).Different plant samples require different and adaptive protein extraction protocols.The leaves of medicinal plant,Baphicacanthus cusia(Nees)Bremek are notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds(especially the secondary metabolites and pigments).This study was aimed to establish a routine procedure for the proteomic analysis of B.cusia leaves,and a new protocol for the protein extraction was developed by optimizing trichloroacetic acid(TCA)/acetone extraction method.The efficiency of this protocol was demonstrated by comparison with 3 published protein extraction methods(chloroform/acetone,Mg/NP-40,Tris-base/acetone).The results showed that the optimized TCA/acetone precipitation extraction method gave a relatively high protein yield(9.263 mg g-1 fresh weight),high-resolution separation,clear protein profiles,the highest proteins spots(1 311 protein spots),and displayed less contamination in 2-DE gels.Therefore,the results suggested that the optimized TCA/acetone method was the most effective among the 4 methods for B.cusia leaves.
Protein extraction is a critical step for two-dimensional electrophoresis (2-DE). Different plant samples require different and adaptive protein extraction protocols. The leaves of medicinal plant, Baphicacanthus cusia (Nees) Bremek are not oriously recalcitrant to common protein extraction methods due to high levels of interfering compounds (especially the secondary metabolites and pigments). This study was aimed to establish a routine procedure for the proteomic analysis of B. cusia leaves, and a new protocol for the protein extraction was developed by optimizing trichloroacetic acid (TCA) / acetone extraction method. The efficiency of this protocol was demonstrated by comparison with 3 published protein extraction methods (chloroform / acetone, Mg / NP-40, Tris-base / acetone). The results showed that the optimized TCA / acetone precipitation extraction method gave a relatively high protein yield (9.263 mg g-1 fresh weight), high-resolution separation, clear protein profiles, the highest protein spots ein spots), and displayed less contamination in 2-DE gels. Herefore, the results suggested that the optimized TCA / acetone method was the most effective among the 4 methods for B. cusia leaves.