Total protein extracts from yeast were prepared as previously described (13 (link)). Protein concentration was determined with a Bradford-based protein assay (Bio-Rad) using bovine serum albumin as standard. A total of 20 μg protein was loaded per lane for SDS–polyacrylamide gel electrophoresis (SDS-PAGE).
For total protein extracts from plants, leaves from 4-week-old rosettes were harvested and snap frozen in liquid nitrogen. Glass homogenizers were used to homogenize the plant material in extraction medium [1 ml/100 mg of fresh weight; 40 mM tris-HCl (pH 6.8), 5 mM MgCl2, 2% (w/v) SDS, and complete protease inhibitor (Roche)]. Insoluble debris was pelleted by centrifugation (5 min, 4°C, 20,000g) before loading equal sample volumes for SDS-PAGE.
For immunoblotting, proteins were transferred onto low-fluorescence polyvinylidene difluoride membranes following SDS-PAGE and probed with antibodies specifically recognizing ESV1, LESV, or green fluorescent protein (GFP). Antisera against Arabidopsis ESV1 and LESV (14 (link)) were used at concentrations of 1:200 for affinity purified anti-ESV1 and 1:3000 for anti-LESV crude serum. YFP-tagged proteins and plant actin (used as a loading control) were detected using commercial antibodies (anti-GFP: Torrey Pines Biolabs, TP401, at a concentration of 1:5000; anti-actin: Sigma-Aldrich, clone 10-B3, at a concentration of 1:10,000).
For total protein extracts from plants, leaves from 4-week-old rosettes were harvested and snap frozen in liquid nitrogen. Glass homogenizers were used to homogenize the plant material in extraction medium [1 ml/100 mg of fresh weight; 40 mM tris-HCl (pH 6.8), 5 mM MgCl2, 2% (w/v) SDS, and complete protease inhibitor (Roche)]. Insoluble debris was pelleted by centrifugation (5 min, 4°C, 20,000g) before loading equal sample volumes for SDS-PAGE.
For immunoblotting, proteins were transferred onto low-fluorescence polyvinylidene difluoride membranes following SDS-PAGE and probed with antibodies specifically recognizing ESV1, LESV, or green fluorescent protein (GFP). Antisera against Arabidopsis ESV1 and LESV (14 (link)) were used at concentrations of 1:200 for affinity purified anti-ESV1 and 1:3000 for anti-LESV crude serum. YFP-tagged proteins and plant actin (used as a loading control) were detected using commercial antibodies (anti-GFP: Torrey Pines Biolabs, TP401, at a concentration of 1:5000; anti-actin: Sigma-Aldrich, clone 10-B3, at a concentration of 1:10,000).
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