Protean 2 apparatus

Total protein content was accessed by Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, United States) according to the kit manufacturer’s protocol, using bovine serum albumin as the standard. In agreement with OIV resolution [Resolution OIV-OENO 452-2012], the protein molecular weight profile of each protein fining agent was acquired by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were prepared in Laemmli Sample Buffer [100 mM Tris–HCl (pH 6.8), 4% (w/v) SDS, 0.01% bromophenol blue (w/v), 0.2% glycerol (v/v), 0.02% β-mercaptoethanol (v/v)]. Proteins were denatured by boiling samples for 5 min at 99°C. SDS-PAGE were performed according to the method of Laemmli (1970) (link). Samples were loaded on 4% stacking gel and run on 12.5% resolving acrylamide gel containing 10% SDS. Gel electrophoresis was performed on a Bio-Rad Protean II apparatus with power supply set at 80 V/gel for the stacking gel and 140 V/gel for the resolving gel. Gels were then stained Coomassie Blue R250 reagent.

As described by Vincenzi et al. (2005 ), a 10% stock solution of sodium-dodecyl sulfate (SDS) was prepared and then added to wine to achieve final concentrations of 0.1%. Samples were gently mixed during 2 min and then heated in a boiling water bath for 5 min. Potassium chloride (KCl) (2 M) was added to each sample to attain a final concentration of 200 mM. Samples were set to incubate for 1 h and KDS-protein pellets were recovered by centrifugation at 14,000 g for 15 min at 4°C (Vincenzi et al., 2005 ). Pellets were further dissolved in 1× phosphate buffered saline, pH 7.4 (PBS buffer). Protein content (%w/w) was further accessed using BCA Assay kit from Pierce. Further, the protein molecular weight profile of each protein fining agent was acquired by SDS-PAGE electrophoresis in concordance with the OIV resolution [Resolution OIV-OENO 452-2012]. Gel electrophoresis was performed on a Bio-Rad Protean II apparatus with power supply set at 100V/gel for the stacking gel and 150V/gel for the resolving gel. Protein samples were equally prepared in Laemmli Sample Buffer (5X) and boiled at 95°C during 5 min. 12.5% polyacrylamide resolving gels were used to process the samples and the gel was afterward stained using Coomassie Blue R250 reagent. The isoelectric point of the protein fining agents and YPE was measured using the Stabino Charge Titration System, measured in triplicates.

The seed storage proteins were extracted from mature kernels or from a part of the kernel without an embryo. The glutenins were extracted, separated by electrophoresis, and visualized according to the International Seed Testing Association standard procedure for SDS-PAGE [25 (link)]. SDS-PAGE was performed using 10% acrylamide concentration and Protean II apparatus (Bio-Rad, Hercules, CA, USA) at 30 mA for 6–10 h and a constant temperature 10 °C. Molecular weight standards, Precision Plus Protein^TM Standards (Bio-Rad) and the HMW-GSs 1B × 6 (cv. Elpa) and 1B × 7 (cv. Genoveva) were used as the molecular weight markers in the electrophoretic mobility evaluation of the novel 1B × 6.5 et al. subunit expressed by the genotype Bagou.