Pageruler unstained low range protein ladder

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using NuPAGE Novex bis-Tris 12-well precast gels (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA), containing 4–12% polyacrylamide, prepared according to the manufacturer’s instructions under reducing and non-reducing conditions in order to study the possible aggregation of proteins using MES (2-(N-Morpholino) ethanesulfonic acid hemisodium salt) buffer. Sample volumes (10 µL) were injected into each well to achieve a protein loading of 0.65 mg/mL. A fixing solution (50% methanol and 10% acetic acid in v/v) was applied to the gels for 2 h before staining with the commercial staining solution SimplyBlue^TM (Thermo Scientific, Vilnius, Lithuania). Thermo Scientific’s PageRuler Unstained Low Range Protein Ladder, with its mixture of purified proteins and peptides in the size range 3.4 to 100 kDa, was used as molecular size markers.

SDS electrophoresis was used to assess the purity of H-NS. Stacking gel contained 4% acrylamide, 0.125M Tris-HCl (pH 6.8), 0.1% SDS. Separating gel contained 12.5% acrylamide, 0.375 M Tris-HCl (pH 8.8), 0.1% SDS. Electrophoresis buffer included 25 mM Tris-HCl, 250 mM glycin and 0.1% SDS (pH 8.3). Ammonium persulfate and TEMED were added to a final concentration of 0.004 and 0.003%, respectively. Sample loading buffer contained 50 mM Tris-HCl (pH 6.8), 100 mM β-mercaptoethanol, 1% SDS, 0.01% bromphenol blue, and 10% glycerol. After sample loading the gels were run at constant current of 25 mA in stacking gel and 45 mA in separating gel. Molecular weight markers used were PageRuler Unstained Low Range Protein Ladder (Thermo Scientific, Lithuania) and Blue Prestained Protein Standard, Broad Range (NEB, United States). Following electrophoresis, the gels were stained in 0.25% Coumassie Brilliant Blue R-250 (Sigma, United States) with 45% ethanol and 10% acetic acid (Khimmed, Russia) and washed in solution containing 40% ethanol and 7% acetic acid.

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.