Purity analysis and 1-D gel separation of peroxisomal proteins. A and B, Leaf peroxisomal proteins (5 μg each lane) were separated on acrylamide minigels. Anti-VDAC immunoblotting (A) and silver staining (B) served to determine the relative contents of mitochondrial VDAC and the P-protein, respectively, while verifying the total amount of loaded proteins in parallel (B). According to the relative content of mitochondrial proteins, leaf peroxisome isolates were classified as of low (lane 1), moderate (lane 2), and high (lane 3) purity. All lanes in each panel are from the same gel. C, Leaf peroxisomal proteins (500 μg) of highest organelle purity were separated on a 10% SDS-PAGE minigel. After brief staining, the gel was cut into 16 slices and proteins were in-gel digested with trypsin and analyzed by LC-MS/MS. The line in S2 separates the stacking and resolving gels.