Preparation of Proteins of Acacia Mangium
We extracted proteins of Acacia mangium according to an altered Hurkman and Tanaka's method.1 Tissue (100 mg) was homogenized with liquid nitrogen and transferred into 200 ^l of preparation buffer (0.7 M sucrose, 0.5 M Trizma base, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2%-mercaptoethanol). The homo-genate was incubated at 4°C for 10 min and had added 50 ^l of phenol solution saturated with water. After ten minutes of incubation at room temperature, it was centrifuged at 8,000 rpm for 10 min at room temperature. The phenol phase was collected and added to 50 ^l of Hurkman and Tanaka's preparation buffer. Following a second 10 min incubation, the extract was again centrifuged at 8,000 rpm for 10 min at room temperature. The phenol phase was collected and added to a five-fold volume of 0.1 M ammonium acetate-methanol. The extract was stored -20°C overnight and centrifuged at 30,000 rpm for 10 min at 4°C. The protein precipitate was washed with 0.1M ammonium acetate-methanol.
The precipitate of the extracted protein was dissolved with the sample buffer described below. After addition of 80 mg of urea, the protein solution was centrifuged at 10,000 rpm for 10 min at room temperature. Two-dimensional electrophoresis was performed.
The pattern of two-dimensional electrophoresis was detected by Coomassie brilliant blue staining. To identify the responsive protein, we compared the electrophoresis pattern of the protein fraction extracted from acid treated Acacia mangium root with that of non-acid treated roots. Using Image Master (Pharmacia), the electrophoresis pattern was analyzed, and the amount of each protein was estimated.
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