The effect of ozone stress (< 10,200 or 400 micrograms m-3) on the protein pattern of Picea abies L. Karst needles was studied by fumigating potted grafts from mature trees. Clonal trees of Norway spruce were fumigated in open-top chambers for one growing season. Proteins, soluble and membrane bound, were extracted from the needles in a four-step procedure: (i) grinding in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenol oxidase and polyvinylpolypyrrolidone (PVP) added to bind phenolic compounds; (ii) precipitation of proteins with acetone at -20 degrees C; (iii) solubilization in a modified lysis buffer; and (iv) dialysis against the modified lysis buffer. Proteins were separated by two-dimensional electrophoresis according to O'Farrell's method. The changes in the silver-stained protein pattern were evaluated by using image processing methods. Electrophoretograms from stressed and nonstressed trees are presented.
by using immobilized pH gradientsAn improved method for two-dimensional electrophoretic separation of acidic proteins extracted from Norway spruce needles is presented. With the use of immobilized pH gradients in the first dimension, this method gave stable separations, with the proteins evenly distributed over the entire gel surface. The number of spots in the electrophoretograms have increased fourfold compared to our earlier separations. The purpose of protein analysis of spruce needles was to investigate stress responses in forest trees using 2-DE as a tool. Our first needle protein study showed that 2-DE was capable of detecting differences in the protein pattern in needles from stressed and nonstressed trees [I] but that our method had potential for improvement. Extraction and separation of soluble and membrane-bound proteins from spruce needles is known to be difficult
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