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Two‐dimensional electrophoresis of acidic proteins isolated from ozone‐stressed Norway spruce needles ( Picea abies L. Karst): Separation method and image processing
Author(s) -
Davidsen Nina B.
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601214
Subject(s) - picea abies , chemistry , chromatography , lysis buffer , lysis , thiourea , bradford protein assay , botany , biochemistry , biology , organic chemistry
The effect of ozone stress (< 10, 200 or 400 μg 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°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.