Nupage novex bis tris 15 well precast gels

The molecular weight distribution of the proteins in samples was evaluated by SDS-PAGE under reducing conditions. 4-12% polyacrylamide NuPAGE® Novex® Bis-Tris 15 well precast gels (Invitrogen, USA) were used according to De Oliveira, Bourlieu, et al. (2016) . Samples were diluted with NuPAGE® LDS sample buffer and distilled water. Reducing conditions were induced by the addition of 0.5 M DL-dithiothreitol to reach a final concentration of 0.05 M. A molecular weight marker (Invitrogen), RGE and porcine pancreatin were used as the controls. Aliquots of 20 μL sample (containing 4 μg of protein) were loaded and the migration was performed at 200 V and 50 mA/gel. Gels were fixed by the addition of the fixing solution (ethanol (30 vol %), acetic acid (10 vol %) and deionized water (60 vol %)). They were then rinsed for 15 min with deionized water before staining with Coomassie Blue with further de-staining by repeated washings with water. Images of the gels were taken and processed using Image Scanner III software (GE Healthcare Europe GbmH, France).

The electrophoretic analyses were performed using 4-12% polyacrylamide NuPAGE® Novex® Bis-Tris 15 well precast gels (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. All samples were diluted 4-fold with NuPAGE® LDS sample buffer and then treated with 0.5 M DL-dithiothreitol and distilled water. Mark 12 Unstained Standard (Invitrogen) was used as a molecular weight (Mw) marker, as reference of the position of the bands. RGE and porcine pancreatin were deposed on the gels as controls, corresponding to the concentrations present in the digestive fluids. Gels were fixed in 30% (v/v) ethanol, 10% (v/v) acetic acid and 60% (v/v) deionized water and were rinsed for 15 min in deionized water before staining with Coomassie Blue. Image analysis of SDS-PAGE gels was carried out using Image scanner III (GE Healthcare Europe GbmH, Velizy-Villacoublay, France). After digitization of gels, the bands were selected and their gray intensity determined by densitometry using the software Image Quant TL™ (GE Healthcare Europe
GbmH, Velizy-Villacoublay, France). Densitometry analyses of the SDS-PAGE gels were used for semiquantification of protein levels. The percentage of each intact protein remaining in the gastric compartment at a given time was estimated in comparison with the undigested human milk.

2.4.2.1. Protein analyses. SDS-PAGE was performed on meal and digesta samples using 4-12% polyacrylamide NuPAGE Novex bis-Tris 15-well precast gels (Invitrogen, Carlsbad, CA, USA). All samples were diluted with NuPAGE® LDS sample buffer and then treated with DL-dithiothreitol and deionized water. Ten µg of meal proteins was loaded into each well, thus taking into account the meal: secretion ratio in order to evaluate the sole impact of proteolysis. Mark 12 Unstained Standard (Invitrogen) was used as a molecular weight (Mw) marker. Gels were fixed in 30% (v/v) ethanol, 10% (v/v) acetic acid and 60% (v/v) deionized water and were rinsed in deionized water before staining with Bio-Safe Coomassie stain (Bio-Rad Laboratories, France). Discoloration of gels was performed with water. Image analyses of gels were carried out using Image scanner III (GE Healthcare Europe GbmH, Velizy-Villacoublay, France). Densitometry on bands was performed by measuring their grey intensity using the software Image Quant TL™ (GE Healthcare Europe183 GbmH, Velizy-Villacoublay, France). This allowed a semi-quantitative analysis of the digesta.