Coomassie brilliant blue g 250 solution

The digestion process was monitored using 16.5% Tricine Mini-PROTEAN^® TGX^™ Precast Protein Gels, 12-well (Bio-Rad, Hercules, CA, USA) gel electrophoresis³⁰ (link).
Aliquots from each phase (oral, gastric, and intestinal) were diluted in sample buffer (10x Tris/Tricine/SDS Running Buffer-BIO-RAD) and vortexed. Then, they were heated (Eppendorf ThermoMixer C) at 95 °C for 5 min and applied to the gel. The gel running condition was at 65 mA for approximately 100 min. After running, the gel was fixed in a fixing solution of methanol, acetic acid, and water (4:1:5 v/v/v). Then, the gel was stained in Coomassie Brilliant Blue G-250 solution (Bio-Rad). The standard low molecular weight marker, the PageRuler Unstained Low Range Protein Ladder (100, 30, 25, 20, 15, 10, 5, and 3.4 kDa) (Thermo scientific^™), was used to monitor the digestion products used.
The evaluation of trypsin inhibitory activity was also performed after simulated in vitro digestion of TTI. It was tested according to the protocol of Kakade et al.³¹ , using Nbenzoyl-DL-arginine-p-nitroanilide (BApNa/1.25 Mm) as substrate. They were collected and analysed at the final time (120 min) of the gastric and intestinal phases.

The pooled aL3Gs were ground using a mortar and pestle after short snap freezing. Lysis buffer was then added and further homogenization was performed using an ultrasonicator. After sonication, the mixture was centrifuged at 12 000 rpm for 15 min at 4 °C. The supernatant was collected and the protein concentration was determined by the Bradford method. Proteins from the lysate were separated by 12% SDS-PAGE gel and later stained with Coomassie Brilliant Blue G250 solution (Bio-Rad, Hercules, CA, USA). After running the SDS-PAGE, the gel was cut into 14 rectangles followed by in-gel digestion.