Criterion tgx stain free gel

Human hippocampus tissue from patients and control samples was lysed with 100 μL lysis buffer containing urea, thiourea, and DTT. After centrifugation at 35.000 rpm for 1 h at 15 °C, extracted proteins were quantified following the Bradford-Protein Assay (Bio-Rad, Hercules, CA, USA) by using a spectrophotometer.
Next, 5 μg of protein per sample were resolved in 4–20% Criterion TGX stain-free gels (Bio-Rad) and electrophoretically transferred onto nitrocellulose membranes using a Trans-blot Turbo transfer system (25 V, 7 min) (Bio-Rad). Equal loading of the gel was assessed by stain-free digitalization and by Ponceau staining. Membranes were probed with rabbit anti-human PLD3 primary antibody (Sigma-Aldrich; 1:250) in 5% nonfat milk and incubated with peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling; 1:2000). Immunoblots were then visualized by exposure to an enhanced chemiluminescence Clarity Western ECL Substrate (Bio-Rad) using a ChemidocMP Imaging System (Bio-Rad). Expression levels of PLD3 were standardized by the corresponding band intensity of GAPDH (Calbiochem; 1:10000).

Myosin heavy chain (MyHC) and actin content was assessed using quantitative gel electrophoresis and immunoblotting, respectively (immunoblots for actin, see the section “Immunoblots”). To extract whole muscle proteins, muscle pieces were homogenized in ice-cold homogenizing buffer (40 μl/mg wet wt) consisting of: 10 mM Tris maleate; 35 mM NaF; 1 mM NaVO₄; 1% Triton X-100 (vol/vol); 1 tablet of protease inhibitor cocktail (Roche) per 50 ml. The protein content was determined using Bradford assay [32 (link)]. Whole muscle homogenates (2.0 μg total muscle mass) were diluted with SDS-sample buffer: 62.5 mM Tris/HCl; 2% SDS (wt/vol); 10% glycerol (vol/vol); 5% 2-mercaptoethanol (vol/vol); 0.02% bromophenol blue (wt/vol) and were loaded onto 4–15% Criterion TGX Stain Free gels (BioRad) together with known amounts of purified recombinant proteins (rabbit myosin and actin, Sigma), the latter allowing a calibration curve to be generated. Gels were imaged (BioRad Stain Free imager) and the density of MyHC was measured by using Image Lab Software (BioRad). When quantifying absolute amounts of MyHC and actin, the density of the relevant band was converted to an equivalent protein amount, according to the calibration curve derived from the pure protein samples run on the same gel. This amount was expressed relative to the muscle mass.

To prepare total protein extract, human hippocampus tissue from patients and control samples was lysed with 100 μL lysis buffer containing urea, thiourea, and DTT (dithiothreitol). After centrifugation at 35,000 rpm for 1 h at 15 °C, protein quantification was determined following the Bradford-Protein Assay (BioRad, Hercules, CA, USA) using a spectrophotometer.
Next, 20 μg of protein per sample was electrophoresed on 4–15% Criterion TGX stain-free gels (Bio-Rad, Hercules, CA, USA) under reducing conditions and transferred onto nitrocellulose membranes using a Trans-blot Turbo transfer system (25 V, 7 min) (Bio-Rad, Hercules, CA, USA). Equal loading of the gel was assessed by stain free digitalization and Ponceau staining. Membranes were probed with mouse anti-human Lamin A + C primary antibody (Abcam, Cambridge, UK) (ab8984; 1:300) in 5% nonfat milk and incubated with peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling, Danvers, MA, USA)(1:2000). Immunoblots were then visualized by exposure to an enhanced chemiluminescence ECL Select™ Western Blotting Detection Reagent (Amersham, GE Healthcare, Chicago, IL, USA) using a ChemidocMP Imaging System (Bio-Rad). GAPDH (Calbiochem, San Diego, CA, USA) (1:10,000) was used as the control in each lane.

Differentially abundant proteins of interest were examined by immunoblotting of pools of protein extracts from all experimental groups and controls from Parts I and II. Protein (10 µg per pool) was fractionated on 12% Criterion TGX Stain-Free gels (Bio-Rad). Gel images were acquired with the Chemidoc system (Bio-Rad) to document equal protein loading among samples. Then, proteins were electrotransferred onto nitrocellulose membranes and probed with following primary antibodies: anti-ATP5F1A (1:1000; #ab151229, AbCam), anti-SDHB [21A11AE7] (1:500; #ab14714, AbCam), anti-PGA3 (1:1000; #PA5-49728, Thermo Fisher Scientific), anti-pepsinogen II/PGC (1:500; #ab135862, AbCam), anti-PDIA3 (internal region; 1:1000; #ABIN3187755, Antibodies-online; Aachen, Germany), anti-GSTP1 (1:1000; #PA5-29558, Thermo Fisher Scientific), and anti-PSME1 (1:1000; #ab14714, AbCam). Primary antibodies were detected with HRP-conjugated goat anti-rabbit and anti-mouse IgG Fc fragment antibodies (1:10,000 dilution; Bethyl Laboratories, Montgomery (TX), USA) and Clarity Western ECL Substrate (Bio-Rad). Blots were imaged with the Chemidoc system.

Western blot analysis was performed on nuclear proteins extracted from liver samples using a commercially available kit (NE-PER™, Thermo Fisher Scientific, #78835). Protein concentration was measured using the Bicinchoninic Acid Kit (Sigma-Aldrich, Saint Louis, MO, USA, #BCA1). Samples were prepared in Laemmli buffer, boiled at 95°C for 5’, loaded into SDS-PAGE precast Criterion TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA) and run under denaturing conditions. Proteins were transferred onto PVDF membranes, blocked with 5% non-fat milk for 45’ at room temperature, followed by incubation with primary antibodies overnight at 4°C (SIRT1: Santa Cruz Biotechnology, Dallas, TX, USA, #SC-15404 1:1000; β-actin: Abcam, Cambridge, UK, #ab8227 1:10000). Membranes were then washed and incubated for 2 h at room temperature with secondary antibody conjugated with HRP (anti-rabbit IgG: Abcam, #ab205722 1:10000). Protein bands were detected using a chemiluminescent substrate (Bio-Rad, #1705061) and imaged onto Kodak film.

The presence of FGB and its possible differential abundance were first investigated by immunoblotting analyses. Within each T group (I and II), proteins extracted from either GC or C tissues were pooled (3 patients per pool). Ten µg of proteins per pool were fractionated on 12% Criterion TGX Stain-Free gels and, after gel image acquisition upon fluorescence excitation with the Chemidoc system (Bio-Rad, Milan, Italy), electrotransferred onto nitrocellulose membranes. Membranes were incubated with the monoclonal antibody anti-FGB [1F9] (1:500; GeneTex, Irvine, CA, USA). Antibody-bound proteins were detected by enhanced chemiluminescence using the Chemidoc system after incubation with ECL HRP-conjugated secondary antibodies (1:10,000 dilution, Santa-Cruz, CA, USA) and reaction with ClarityTM Western ECL Substrate (Bio-Rad, Milan, Italy). Because of the lack of universal house-keeping genes to be used as sample loading control in GC [62 (link)], image of the stain-free gel acquired before its transfer was used as control for equal protein loading among samples.