Gel doc universal hood 2

The protein composition of both fresh and digested EH-WPC and MP-WPC was evaluated by sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE) on NuPAGE^TM 4–12% bis-tris midi protein gel (ThermoFisher Scientific, Amsterdam, The Netherlands) performed as previously reported [26 (link)]. Equal levels of protein per sample were loaded, as determined by bicinchoninic acid (BCA) reaction [27 (link)]. Analysis was performed under reducing (with 2-mercaptoethanol (2ME)), and also under non-reducing conditions, to evaluate the protein composition of aggregates held together by disulfide bridges. Proteins and peptides were identified using the PageRuler^TM plus protein ladder (10–250 kDa, #26619, ThermoFisher Scientific, Amsterdam, the Netherlands), and included bovine serum albumin (BSA) 69 kDa, immunoglobulin G heavy chain polypeptide (IgG HC) 53 kDa, β-casein (βCAS) 24 kDa, β-lactoglobulin (βLG) 18 kDa and α-lactalbumin 14 kDa. Gels were scanned using Gel Doc Universal Hood II (Bio-rad, Hercules, CA, USA) and densitometric analysis was performed in Quantity One version 4.6.9 (Bio-Rad, Hercules, CA, USA). Each peak intensity displayed on the densitogram corresponds to the amount of soluble proteins that migrated through the gel to their specific protein size. Only densitograms from samples run on the same gel were compared.

For each set of samples, equal volume containing 10 μg or 20 μg of protein sample was loaded and the proteins were separated by SDS-PAGE and subsequently transferred to an activated PVDF membrane (MDI Membrane Technologies, Ambala, India). The blots were first incubated in a blocking solution (5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST); pH 7.4) followed by overnight incubation in a primary antibody prepared in the blocking solution, at 4°C. The blots were then washed for 3×10-min in TBST and probed with anti-rabbit or anti-goat secondary antibody conjugated with horse-radish peroxidase (Vector Laboratories) for one hour at room temperature, followed again by 3×10-min washing in TBST. The signals were detected using enhanced chemiluminescence (ECL; Millipore, Billerica, USA) with GelDoc (Universal Hood II, Bio-Rad, Hercules, USA). Densitometric analysis was performed with ImageJ software (National Institutes of Health, USA). The grayscale image was ‘‘inverted” and the background was uniformly subtracted using rolling ball radius method. The signal of each protein was normalized with the loading control, and expressed as integrated optical density (OD).

Ribonucleic acid (RNA) from each liver was isolated using Tri reagent (Sigma). DNA contaminants were removed by TURBO DNAse treatment using the DNA removal kit from Thermo Fisher Scientific (Madrid, Spain). RNA was quantified by absorbance at 260 and 280 nm using a SPECTROstar^Nano and software version 2.12 (BGM LABTECH, Ortenberg, Germany). The A260/280 ratio was higher than 1.7 in all samples. The integrity of the 28S and 18S ribosomal RNAs was verified by 1% agarose gel electrophoresis. Ethidium-bromide stained gels were exposed to UV light and images were captured using a Geldoc Universal Hood II and the software Quantity One version 4.6.6 (BioRad, Madrid, Spain).