## 2 Materials and Methods
### Protein and Amino Acid Analysis of Soybeans and Pea Beans
#### 2.3.3 Protein Electrophoresis  
The general principle of the method is described in the European Pharmacopoeia [41; 42; 43; 44]. Preparation of the solubilization buffer and sample preparation: a 2.5 mL concentration gel buffer solution, 2.0 mL glycerol, 4.0 mL sodium dodecyl sulfate (SDS) 10% solution, 0.5 mL Coomassiee Brilliant Blue G-250(r) 0.1% solution, 1 mL Milli-Q(r) ultrapure water were added to a test tube and homogenized. Then, 2-mercaptoethanol ethanol 1:20 (\(v/v\)) was added before use. It has an antioxidant role. The test solutions were osbtained from 60 mL of previously prepared sample solution and 60 mL of solubilization buffer. They were shaken and kept in the water bath at 95 \({}^{\circ}\)C for 5 min to denature the proteins. 1. The 30% acrylamide-bisacrylamide solution was prepared as follows: 29.2000 g of acrylamide and 0.8000 g of N,N\({}^{\prime}\)-methylenebisacrylamide were weighed and were introduced into a 100 mL volumetric flask. They were dissolved in Milli-Q(r) ultrapure water and made up to volume with the same solvent. Then, they were filtered and stored in brown glass vials in the refrigerator for 30 days. 2. Preparation of the 10% sodium dodecyl sulfate (SDS) solution: 10 g of sodium dodecyl sulfate was weighed and placed in a 100 mL volumetric flask. It was dissolved in Milli-Q(r) ultrapure water and made up to volume with the same solvent. The solution was stored at room temperature. 3. Preparation of gel buffer for separation: 18.1500 g of Trizma base(r) (tromethamine) was weighed and placed in a Berzelius beaker. Then, it was dissolved into Milli-Q(r) ultrapure water, and the pH was adjusted to 8.80 using the Mettler Toledo FiveEasy pH F20 pH meter with 6 N HCl solution. The content was transferred to a 100 mL volumetric flask and brought to the mark with the same solvent. It was stored in the refrigerator. The final concentration of Tris was 1.5 M.
4. Preparation of the gel buffer for concentration: 6.0000 g of Trizma base(r) was weighed and placed into a Berzelius beaker. Then, it was dissolved in Milli-Q(r) ultrapure water, and the pH was adjusted to 6.80 using the Mettler Toledo FiveEasy pH F20 pH meter with 6 N HCl solution. The contents were transferred into a 100 mL volumetric flask and made up to the mark with the same solvent. It was stored in the refrigerator. The final concentration of Tris was 0.5 M.
5. Preparation of 10% ammonium persulfate (APS) solution: 0.1000 g of ammonium persulfate was weighed using the analytical balance and dissolved in 1 mL of Milli-Q(r) ultrapure water. 6. Preparation of migration buffer pH 8.3:3.0300 g of Trizma base(r) and 14.4000 g of glycine were weighed on the analytical balance. They were placed in a 1000 mL volumetric flask. It was dissolved in ultrapure Milli-Q(r) water. Then, 10 mL of 10% SDS solution was added and made up to the mark with the same solvent. No pH adjustment was made. 7. Preparation of fixing solution: 12.0000 g of trichloroacetic acid was weighed and placed in a 100 mL volumetric flask. It was dissolved in Milli-Q(r) ultrapure water and made up to a volume with the same solvent. 8. Preparation of Coomassieie staining solution: 0.1800 g of Coomassieie Brilliant Blue G-250(r) was dissolved in 45 mL of methanol. In total, 10 mL glacial acetic acid and 45 mL Milli-Q(r) ultrapure water were added. 9. Preparation of the bleaching solution: the solution was prepared with a volumetric methanol/acetic acid/ultrapure water ratio of 40:10:50. 10. Preparation of anti-fade solution: a solution of 2% acetic acid in ultrapure water was prepared. 11. Tris-glycine pH 8.3 buffer was prepared according to the procedure presented for protein extraction. 1. The formation of gels Plates of 10 mm thickness were selected and assembled in dedicated supports resting on an absorbent sponge for the polymerized separation gel. Ultra-pure water, 30% solutionof acrylamide-bisacrylamide, Tris tampon and 10% SDS solutions were mixed.