SDS-PAGE Separation of Wheat Proteins

Electrophoretic runs were performed according to the study by Ruiz et al. (2016) (link) with minor modifications. Proteins (40 mg lane^–1) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using The Mini Protean TETRA apparatus (Bio-Rad Laboratories, Italy). The molecular mass standard was the Neobiotech (Nanterre, France) Elite Prestained Protein Ladder (6.5–270 kDa). Samples were dried (RapidVap Evaporator Labconco) under vacuum and resuspended in 30 ml MilliQ water, and thus, sample buffer was added [final concentrations 2% SDS, 10% glycerol, 200 mM 2-mercaptoethanol, and 0.01 mg/ml bromophenol blue in 63 mM Tris-HCl (pH 6.8)]. Samples were boiled for 5 min prior to loading. Gels were fixed at room temperature in methanol/glacial acetic acid/water (50/5/45, v/v) for 20 min and then in 50% methanol for 10 min. After fixing, gels were stained with Bio-Safe Coomassie blue (Bio-Rad, Milan, Italy) as described in the instruction protocol.
Gliadins were subdivided into two classes (ω- and α-, γ-) based on their molecular weight, whereas glutenins were subdivided into HMW-GS and LMW-GS (De Santis et al., 2017 (link)). Protein profiles were analyzed using the Azurespot Pro software (Azure Biosystems, Dublin, CA, United States).