Protein Extraction and Quantification Protocol

Total protein was extracted from samples collected in both 2010 and 2011 growing seasons by the previously described protocol of Wang et al. (2006) (link) with slight modifications. Samples (0.5 g) were first homogenized in liquid nitrogen in the presence of polyvinylpolypyrrolidone (PVPP). The protein pellets obtained were dissolved in SDS-PAGE sample buffer containing 0.15 M Tris (pH 6.8), 1.2% SDS, 30% glycerol, 2.14 M β-mercaptoethanol (Sigma-Aldrich, St Louis, MO, United States). Extracted proteins were quantified by band intensities confirmed by fractionating on 10% SDS-PAGE gel, and staining with Coomassie protein staining buffer (0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to PROTEAN nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) using the Mini-Protean Transfer system (Bio-Rad Laboratories, Hercules, CA, United States). Immunoblot assays are as previously described (Acheampong et al., 2015 (link)). Band intensities were analyzed using ImageJ 1.48v software (Schneider et al., 2012 (link)).