Precision plus protein all blue standard

Whole raw milk and raw cream were provided by a local supplier (Quebec City, QC, Canada) and skim milk powder (SMP) was obtained from Agropur (Quebec City, QC, Canada). The thermophilic yogurt culture YC-X11 Yo-Flex^® (Chr. Hansen A/S, Hørsholm, Denmark) was composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus. Analytical-grade sodium hydroxide for the preparation of 0.1 M of NaOH was obtained from Fisher Chemical (Ottawa, ON, Canada). Mini-PROTEAN TGX Stain-Free Gels (12%, 15-well comb, 15 µL), 2× Laemmli sample buffer, native sample buffer, Precision Plus ProteinTM All Blue Standards, 10× Tris/glycine/sodium dodecyl sulfate (SDS) buffer, and 10× Tris/glycine buffer were all obtained from BioRad (Hercules, CA, USA). 2-Mercaptoethanol was provided by Sigma-Aldrich (St. Louis, MO, USA). Methanol was obtained from Fisher Chemical (Ottawa, ON, Canada) and glacial acetic acid from Anachemia (Radnor, PA, USA). Fast Green FCF and Nile Red were obtained from Sigma-Aldrich (Oakville, ON, Canada).

Proteins were analyzed by Western blotting. Each protein extract was mixed with one volume of Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and boiled for 5 min. Precision Plus Protein^TM All Blue Standards (Bio-Rad Laboratories) were used as protein size markers. Equal amounts of protein (3 µg) were separated on 4-20% SDS-PAGE gels (Criterion TGX precast Gels, Bio-Rad) and electrotransferred to PVDF membrane using an iBlot2 transfer system (Thermo Fisher Scientific). The membranes were blocked with StartingBlock T20 blocking reagent (Thermo Fisher Scientific) and incubated overnight at 4 ℃ with 1:100 dilutions of antibodies to the N-terminal of dystrophin (NCL-DYSB, Leica Biosystems, Wetzler, Germany), the C-terminal of dystrophin (ab15277, abcam, Cambridge, UK) and a 1:1000 dilution of antibody to vinculin (ab129002, abcam) as a loading control. After washing, the membranes were incubated with a 1:20,000 dilution of secondary HRP-coupled antibody to rabbit for ab15277 and ab129002 (GE Healthcare Life Sciences) and to mouse for NCL-DYSB (GE Healthcare Life Sciences). After washing, the membranes were processed for enhanced chemiluminescence detection using Luminata Forte Western HRP substrate (EMD Millipore, Billerica, MA, USA). Immunoreactive proteins were visualized by Amersham Imager 680RGB (GE Healthcare Life Sciences).

Protein in sample buffer was denatured under reducing conditions at 57°C for 15 min. Samples and prestained molecular weight marker (Precision Plus Protein All Blue Standards, Biorad) were separated on polyacryamide gels (5, 12% gels; or 4-15% gradient gels) in 25 mM Tris Base, 192 mM glycine, 0.1% SDS. Separated proteins were blotted on polyvinylidene fluoride (PVDF) membrane [Immobilon-P Transfer membrane, Millipore; transfer buffer: 25 mM Tris base, 192 mM glycine, 20% (v/v) methanol, with or without 0.1% (w/v) SDS]. Coomassie staining of gels was performed to check for efficiency of the transfer. Immunostained bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific) and a Fusion Fx7 Peqlab bioimager. Quantitation of band intensity was performed with Image J 1.46 (National Institute of Health). For quantification integrated density of specific bands was normalized against loading control. Unspecific bands and Coomassie-stained membranes were used as loading control. Quantification of gel or blot intensities was performed with data obtained within a linear range of exposure.

Twenty microliters of the protein sample was mixed with 5 μL of loading buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% (w/v) SDS, 0.1% bromophenol blue, 10% (v/v) glycerol). The mixture was incubated at room temperature for 30–60 min, and then loaded on polyacrylamide gel (with 15% w/v acrylamide) in duplicates. After electrophoresis, the gel was cut into two halves, each with a lane of the sample and molecular weight standard (Precision Plus Protein All Blue standards, Biorad, Hercules, CA, USA); one half was stained with Coomassie Brilliant Blue R 250 while the other was tested for antibacterial activity by the method described earlier [53 (link)]. For the latter, the gel was fixed immediately in a solution of 20% isopropanol and 10% acetic acid for 2 h, washed in distilled water for 6 h, placed in a sterile Petri dish and overlaid with 15 mL of soft MH agar (0.5×) seeded with the test strain (G. stearothermophilus strain 10), and incubated at 60 °C for 12 h for development of the inhibition zone (Scheme 1).

Aliquots of muscle homogenate (25 μg per sample) were solubilized in Laemmli sample buffer (BioRad Laboratories, Inc., CA, USA), denatured by boiling at 100°C for 5 min and separated by SDS-PAGE. Molecular weight standards (Precision Plus Protein All Blue Standards, BioRad) were also run on each gel to identify the proteins molecular weights. One-dimensional gels were stained by Coomassie Brilliant Blue R-250 (BioRad), and the stained gel images were semiquantitatively analyzed using the Image Studio Lite 3.1.4 software for Macintosh (LI-COR Biosciences, NE, USA).
Bands of interest were manually excised and sent for identification by peptide mass fingerprint to the Inbiotec S.L. (León, Spain) proteomics laboratory, where the samples were processed and analyzed with a 4800 Proteomics Analyzer matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometer (ABSciex, MA, USA) according to the previously described methods of Oliván et al [25 ]. A database search on Mascot Generic Files combining MS and MS/MS spectra was performed using Mascot v 2.2 from Matrix Science through the Global Protein Server v 3.6 (ABSciex). When the Mascot score was greater than 85 points, the identified protein was considered a valid candidate.