Mini protean 2 dual slab cell system

Native-PAGE or "non-denaturing" gel electrophoresis was performed to evaluate β-Lg structures integrity during in vitro digestion. The electrophoresis analysis was carried out using Mini-Protean II dual slab cell system equipped with a PAC 300 power supply (Bio-Rad Laboratories, Hercules, CA, USA), according to Madalena et al. (2016) (link) methodology. The resolving and stacking gel contained 12.5 % and 3.5 % of polyacrylamide, respectively. The gels were stained and maintained in Coomassie Brilliant Blue (R-250) solution, composed by 50 % methanol and 10 % acid acetic, and maintained overnight with gentle agitation. Afterwards, gels were destained with solution constituted by 30 % methanol and 7 % acetic acid. Standard marker protein PageRuler Unstained Broad Range Protein Ladder (Thermo Scientific) was used to identify samples by their molecular weight.

In order to evaluate the heat treatment effects on nanohydrogel kinetic formation, patterns of Lf, GMP and Lf-GMP mixtures samples were analysed using Native-PAGE or "nondenaturing" gel electrophoresis. Native-PAGE analyses were carried out using the Mini-Protean II dual slab cell system equipped with a PAC 300 power supply (Bio-Rad Laboratories, Hercules, CA, USA). The resolving and stacking gel contained 10 and 4% of polyacrylamide, respectively. The gels were stained with silver nitrate methodology (Chevallet, Luche, & Rabilloud, 2006) . Standard marker proteins PageRuler Unstained Broad Range Protein Ladder from Thermo Scientific, was used to identify molecular weight of samples.

In order to evaluate the heat treatment effects on denaturation and aggregation patterns of whey protein subunits, unheated and heated WPI samples were analyzed using Native-PAGE or "nondenaturing" gel electrophoresis. Native-PAGE analyses were carried out using the Mini-Protean II dual slab cell system equipped with a PAC 300 power supply (Bio-Rad Laboratories, Hercules, CA, USA). The resolving and stacking gel contained 10 and 3.7% of polyacrylamide, respectively. Whey protein samples were mixed with twice their volume of a non-reducing loading buffer of TRIS 0.5 mol L À1 at (pH 6.8, 50% glycerol and 0.02% bromophenol blue). The gels were maintained overnight in 50% ethanol and 10% of acetic acid solution, stained with Comassie Brilliant Blue (R250) solution and destained with a 5% ethanol and 7.5% acetic acid solution. The integrated intensities of whey protein bands were determined using the Bio-Rad software "Bio-Rad Quantity One", associated with a GS-800 Densitometer (Bio-Rad Laboratories, Hercules, CA, USA). The quantity of each protein was determined as a percentage of that in the unheated samples (Anema & Li, 2003) .

Plantaris muscles were extracted on ice for 60 minutes in four volumes of extracting buffer (pH 6.5), as previously described. 41 After centrifugation, the supernatants were diluted 1:1 (v/v) with glycerol and stored at À20 C. Myosin heavy chain isoforms were separated onto 8% polyacrylamide gels, which were made in the Bio-Rad (Marnes-la-Coquette, France) mini-Protean II Dual slab cell system, as described previously. 42e44 The gels were migrated for 31 hours at 72 V (constant voltage) at 4 C. For the quantification of the relative concentration of myosin heavy chain and actin, 1 mg of total proteins was loaded and 12% polyacrylamide gels were used. 45 The silver-stained gels were scanned using a video acquisition system, and bands were quantified by densitometric software version 381472 (Multi Gauge; Fujifilm, Asnières, France).