Computational protocol: Aging Affects the Transcriptional Regulation of Human Skeletal Muscle Disuse Atrophy

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Protocol publication

[…] From each muscle biopsy 150 cryosections (10 µm) were homogenized in a micro vial containing 1 silicium carbide crystal, 5 steel beads (2.3 mm) and 250 µl ice-cold homogenization buffer (50 mM Tris-base, 1 mM EDTA, 1 mM EGTA, 10 mM beta-glycerophosphate, 50 mM sodium fluoride, 0.5 mM sodium orthovanadate, 0.1% v/v, Triton-X, 0.1% v/v mercaptoethanol and protease inhibitor (Complete, Roche, Basel, Schwitzerland), pH 7.5) using a FastPrep-24 (MP Biomedicals, Solon, OH, USA) homogenizer. Laemmli buffer was added and protein concentrations were determined with the EZQ Protein Quantitation Kit according to the manufacturer's protocol (Molecular Probes, Eugene, OR, USA). Then, samples were heated at 90°C for 4 min, shortly vortexed and spun in a microcentrifuge and equal amounts (10 µg/4 µl) were separated by SDS-PAGE using a 4–12% Bis-Tris gel (Criterion, Bio-Rad, Hercules, CA, USA) at 200 V for 1 h. Gels were blotted (Trans-blot cell, Bio-Rad, 400 mA, 2 h) to polyvinylidene difluoride membranes (Amersham Hybond LFP, GE Healthcare, Buckinghamshire, UK), which were blocked for 30 min with 20% Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE, USA) in phosphate-buffered saline, incubated overnight at 4°C with primary antibody, incubated for 1 h in fluorophore-conjugated with secondary antibody and visualized with the Odyssey Infrared Imaging System (Li-Cor Biosciences). Total and phosphorylated protein pairs were detected simultaneously on the same membrane. Band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). Total and phospho (serine 473) Akt primary antibodies (Cell Signaling Technology, Danvers, MA, USA, no. 2920 and 4060) were diluted 1∶2,000 and actin (Sigma, Saint Louis, MO, USA, no. A2066) primary antibody was diluted 1∶10,000. Due to low tissue availability n equals 3 in each age group for the measurement of total and phospho Akt. Western blot analysis for LC3B, as well as S6 ribosomal protein and phospho-S6 ribosomal protein (Ser235/236) were performed on frozen tissue homogenized in 10 volumes (wt/vol) of ice-cold buffer (300 mM Sucrose, 1 mM EDTA, 10 mM NaN3, 40 mM tris-base and 40 mM histidine at pH 7.8 with protease inhibitors, #05892791001, Roche Inc.) using a 1 ml glass homogenizer with a glass pestle (Kontes Glass Industry, Vineland, NJ). Protein content in the muscle homogenate was measured in triplicates using a standard kit (Pierce BCA protein reagent no. 23225, Pierce Inc.). Samples were heated at 90°C for 4 min, shortly vortexed and spun in a microcentrifuge and equal amounts (20 µg) were separated by SDS-PAGE using a 4–15% Tris/glycine gel (Miniprotean TGX, BioRad, Hercules, CA, USA) at 200 V for 35 min. Gels were blotted (Trans-blot cell, Bio-Rad, 250 mA, 1 h) to polyvinylidene difluoride membranes (Immun-Blot PVDF, 0.2 µm, BioRad, Hercules, Ca, USA), which were blocked for 60 min with 5% Blotting–Grade Blocker (#170-6404, BioRad, USA) in phosphate-buffered saline with 0.05% Tween 20. Membranes were subsequently incubated overnight at 4°C with primary antibody LC3B (1∶1000, #2775, Cell Signaling Technology) as well as S6 ribosomal protein (1∶1000, #2217, Cell Signaling Technology Inc.) and phospho-S6 ribosomal protein (Ser235/236) (1∶2000, #4858, Cell Signaling Technology Inc.). Membranes were subsequently washed and incubated for 1 h with HRP-conjugated secondary antibody (Immun-Star Goat Anti-Rabbit (GAR)-HRP Conjugate, #170-5046, BioRad, Hercules, Ca, USA) and visualized with Immun-star western kit (#170-5070, BioRad, Hercules, Ca, USA). After detecting the phosphorylated protein, the membrane was stripped and the total protein was detected. Band exposure and visualization were quantified using ChemiDoc XRS with Image Lab Software (Bio-Rad Laboratories, Inc.) […]

Pipeline specifications

Software tools ImageJ, Image Lab
Application Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Muscular Diseases