Himark pre stained protein standard

Proteins were size-fractionated using precast 4–12% gradient NuPAGE Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes (Whatman). Membranes were blocked in either 5% (w/v) non-fat milk powder or 5% (w/v) BSA before probing with the specified antibodies. Primary antibodies were used at the following titres; 1:1600 for rabbit monoclonal anti-sacsin (Abcam), 1:5000 for mouse monoclonal anti-HSP70 (Sigma) 1:5000 for mouse monoclonal anti-Lamp2 (Santa Cruz), 1:3000 for rabbit polyclonal anti LC3 (Abcam) 1:10 000 for mouse monoclonal anti-β-actin (Sigma), 1:5000 for rabbit polyclonal anti-GAPDH (Abcam), 1:1000 for mouse monoclonal anti-p62 (Abcam), and 1:3000 for rabbit polyclonal anti-LC3 (company). Immunoreactive products were visualized and quantified, after labelling with species-specific infrared secondary antibodies (1:5000, LI-COR Biosciences, UK), using the Odyssey imaging system (LI-COR). Apparent molecular masses were estimated using the Novex Sharp Pre-stained Protein Standard (Life Technologies) or HiMark Pre-stained Protein Standard (Life Technologies) and the band sizing application in Odyssey software (LI-COR Biosciences). Images of the immunoblots were also quantified using the Odyssey software.

The molecular mass distribution in each of the two fibroins was investigated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). A Novex^® XCell Sure Lock™ Mini-Cell system (Life Technologies Inc, Carlsbad, CA, USA) and an EPS-250 Series II Power Supply unit (CBS Scientific Company Inc, San Diego, CA, USA) were employed. The fibroin membranes (prepared as described in the previous section) were solubilized either in a solution of lithium bromide (the BMSF membranes) at 60 °C, or in neat calcium nitrate at 105 °C (the APSF and BMSF/APSF blended membranes), and the resulting solutions were dialyzed and filtered. Each solution was mixed with both NuPAGE^® sample preparation reagent and NuPAGE^® sample reducing agent, and heated at 70 °C for 10 min. A volume of each solution containing about 50 μg protein was loaded into a 1-mm thick 3–8% NuPAGE^® Novex^® Tris–Acetate gel in NuPAGE^® Tris–Acetate SDS Running Buffer. The gels were run at a voltage of 150 V for 1 h together with a HiMark™ Pre-stained Protein Standard (Life Technologies). The gel was then washed in three 5-min stages with distilled water, and soaked in SimplyBlue™ SafeStain solution containing Coomassie^® G-250 stain for 1 h under gentle stirring. The resulting gel was washed in distilled water for 1 h and then photographed.

All samples were boiled in 1 × Laemmli buffer and subsequently separated by gel electrophoresis in NuPAGE 3–8% Tris–acetate SDS polyacrylamide gradient gels (Invitrogen) together with Spectra Multicolour High Range Protein Ladder (Thermo Fisher) and HiMark Prestained Protein Standard (life technologies) as range indicators. Separated proteins were transferred to nitrocellulose membranes. After blocking with 5% block milk, membranes were incubated with corresponding primary and HRP-bound secondary antibodies in 5% block milk. Visualization was performed using ECL reaction (Western Blotting Luminol, Santa Cruz Biotechnology) on X-Ray Film (Fuji Medical Super RX).

Proteins were extracted from myotubes by replacing culture medium with NHC lysis buffer (4 M urea, 125 mM Tris pH 6.8 and 4% SDS) supplemented with 1X Complete Protease Inhibitor Cocktail Tablets (Roche). Samples were and left on ice for 10 min after which they were collected, boiled for 3 min and centrifuged at 14000×g for 10 min at 4 °C. Protein quantification was done with the Pierce BCA protein assay (Thermo Fisher Scientific). 50 μg of protein samples were run on a precast NuPAGE Novex Tris- Acetate 3–8% gradient gel (Invitrogen) according to manufacturer’s instructions. 10 μl of the HiMark pre-stained protein standard (Life Technology) and the Odyssey One-Color protein molecular weight marker (Licor) were included as a reference molecular weight to analyse the protein of interest. The gel was run on ice at 75 V for 45 min to allow dystrophin to slowly enter the gel and then at 150 V for 2 h and 15 min.

Representative samples from each antibody at each timepoint (0, 2, and 4 weeks) were run on an Invitrogen NuPAGE 3%–8% Tris-Acetate Gel. Protein samples were diluted from 10 mg/ml down to 0.33 mg/ml with water. To each of the 0.33 mg/ml samples, 5 µl of loading buffer (NuPAGE LDS Sample Buffer 4X, Invitrogen) were added, yielding 1:3 sample:loading buffer, with a final antibody concentration of 0.25 mg/ml 10 µl of the 0.25 mg/ml antibodies were added into individual wells. 15 µl of the ladder (HiMark^TM pre-stained protein standard, Invitrogen) were added into well 1. The gel was run under the following conditions: 150 V, 50 mA, 5 W for 1 h on a PowerEase500 electrophoresis system (Invitrogen). Upon completion of the run, the gel was washed 3 times with water, shaking each time for 5 min. Then the gel was washed with SimplyBlue SafeStain (Invitrogen) for 1 h with shaking and with water for 1 h with shaking. The gel was imaged using a FluorChem M Imaging System (Protein Simple).