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Powdered fruit or peel tissue (0.2 g) was placed in a 2.0 mL microfuge tube and the protein precipitated at 2207C for 45 min with 1.75 mL of a precipitation solution (10%TCA in ice-cold acetone). The precipitated protein was centrifuged for 10 min at 10 0006g at 297C. The supernatant was discarded and the pellet rinsed with 2 mL of ice-cold acetone and stored at 2207C for 1 h to remove residual TCA then further centrifuged for 15 min at 10 0006g at 2207C. Acetone rinses were repeated until a white pellet was obtained. The protein pellet was dried under vacuum for 5 min and then dissolved in varying volumes of buffer containing 5 M urea, 2 M thiourea, 2%CHAPS, 2% N-decyl-N,N-dimethyl-3- ammonio-1-propanesulfonate, 20 mM DTT, 5 mM tris(2- carboxyethyl) phosphine hydrochloride, and two carrier ampholytes 0.5%pH 4–6.5 and 0.25%pH 3–11 nonlinear.
