6-week-old A. thaliana Col_0 plants were sprayed with 300 µM methylviologen (MV) and 0.1% (v/v) Tween-20 as control, respectively. After 3 h the plants were harvested and immediately frozen in liquid nitrogen and ground to a fine powder. Proteins were isolated and used for the separation in the first dimension with Immobiline Dry Stripes (pH range 3–10 NL, 18 cm, GE Healthcare, Uppsala, Sweden) [21]. 250 µg protein were dissolved in 340 µL rehydration buffer (0.01% ampholyte; 0002% (w/v) bromophenol blue) and applied to the Immobiline Dry Stripe. The rehydration and focusing consisted of the following steps: 1 h 0 V, 12 h 30 V, 2 h 60 V, 1 h 500 V, 1 h 1000 V, and finally 8000 V for as long as needed to reach 42,000 Vh. Separation in the second dimension was done with 12% non-reducing SDS-PAGE. Afterward, the gel was blotted to nitrocellulose membrane and subjected to Western blotting with PRX-IIE antibody and peroxidase-labeled secondary antibodies. Detection was done with ECL Substrate (GE Healthcare, Chicago, IL, USA) and X-ray films.
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