Westernbreeze chromogenic western blot immunodetection kit

Total and phosphorylated mTOR were evaluated by western blotting analysis. Samples of colon were weighed and homogenized in buffer containing 20 mmol/L Tris-HCl, pH 7.4, 120 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L ethylene glycol bis (2-aminoethylether) tetra-acetic acid, 50 mmol/L β-glycerophosphate, 50 mmol/L NaF, 0.3% 3-(3-cholamidepropyl) dimethylammonio-1-propanesulphonate, 1 mmol/L dithiothreitol, 4 mg/mL leupeptin, and 4 mg/mL aprotinin using a tissue homogenizer. The remaining homogenate was centrifuged at 12,000 g for 20 min at 4°C. Cell lysates containing equal amounts of protein were separated on 4–12% tris-glycine polyacrylamide gels and then electrophoretically transferred to nitrocellulose membrane. Membranes were incubated with anti-mTOR (L27D4), antiphospho-mTOR (Ser2448), and phospho-p70 S6 kinase (Thr389) specific antibodies (Cell signaling Technology). Then immunoreactive bands were detected using a Western Breeze Chromogenic Western Blot Immunodetection Kit (Invitrogen, Carlsbad, CA, USA).

Solubilization screens were conducted in 5 ml total volume mixtures with membranes diluted to 1 mg/ml and solubilized in buffer containing 300 mM NaCl, 1% (w/v) glycerol, and 20 mM Tris-HCl pH 8.0. The concentration of detergent added varied depending on type: 1.5% (w/v) DDM, 1.4% (w/v) DM, and 5% (w/v) OG. After incubation for 1 h at 4°C, soluble and insoluble fractions were separated by centrifugation at 35,000 rpm for 30 min.
Conventional protein detection methods, such as SDS-PAGE gel electrophoresis and Western blot analysis, were used to analyze the soluble fractions for protein yield and efficiency of extraction. Solubilized ClC-rm1 fractions in detergent were analyzed by NUPAGE 4–12% Bis-Tris gel electrophoresis (Invitrogen) and 1% (v/v) MES as running buffer (Invitrogen) and visualized by SimplyBlue SafeStain (Invitrogen) or Silver stain. Western blot was carried out according to the manufacturer’s instructions using the WesternBreeze Chromogenic Western Blot Immunodetection Kit (Invitrogen) and His tag antibody as primary antibody (ABGENT). The secondary antibody was an alkaline phosphate conjugated, affinity purified, anti-specific IgG (Invitrogen).

Washed THP-1 cells (5 × 10⁵) were lysed on ice for 15 min using 100 μL of lysis buffer from a WSE-7420 EzRIPA Lysis kit (ATTO Corporation, Tokyo, Japan) and the supernatants were processed by centrifuging at 14,000× g for 5–15 min, according to manufacturer’s protocol. The protein concentration was determined by the method of Bradford (Bio-Rad Protein Assay, Hercules, CA, USA). Twenty micrograms of protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard 15% or 5–20% gradient gels, followed by Western blot analysis using an iBlot dry blotting system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Antibodies O-17 (1:100 dilution; IBL, Gunma, Japan), 34E3 (1:100 dilution; IBL), 10A16 (1:200 dilution; IBL), and ab8448 (1:1000 dilution; Abcam, Tokyo, Japan) were used as primary antibodies (1 μg/mL each). The bands were detected using a WesternBreeze Chromogenic Western Blot Immunodetection Kit (Invitrogen). In some experiments, the bound primary antibodies were reacted with horseradish peroxidase-conjugated mouse or rabbit anti-mouse IgG (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) diluted 1:20,000 and visualized using a TMB Membrane Peroxidase Substrate system (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) according to the manufacturer’s protocol.

Total proteins extracted from the cells in the different experimental conditions were quantified, as reported previously [48 (link)]. Forty µg of total proteins were subjected to electrophoresis on NuPAGE® 4%–12% Bis-Tris Gel (Invitrogen, Life Technologies; 200 V, 40 min) and blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen, Life Technologies; 30 V, 1 h). The membranes were incubated with mouse monoclonal anti-α-sma (1:1000; Abcam), rabbit polyclonal anti-Cx43 (1:2500; Chemicon), and mouse monoclonal anti-Cx26 (1:500; Sigma) overnight at 4 °C. Immunodetection was performed according to the Western Breeze® Chromogenic Western Blot Immunodetection Kit protocol (Invitrogen, Life Technologies). The same membranes were subjected to the immunodetection of the expression of α-tubulin (rabbit polyclonal anti α-tubulin, 1:1000; Merck, Milan, Italy), assumed as control invariant protein. Densitometric analysis of the bands was performed using ImageJ 1.49v software (NIH, https://imagej.nih.gov/ij/), and the values normalized to control.

HepG2 nuclear extracts (300 μg) were incubated with 15 μl of protein A/G-agarose beads and 8 μg anti-H1.2 or Aly antibodies as previously described (17). Pre-immune IgG was used as a negative control. Eluted proteins were separated by SDS-PAGE and electrophoretically transferred to a polyvinylidine difluoride membrane (Millipore). Proteins were detected with the indicated antibodies in a dilution of 1:100 to 1:500 with a Western Breeze Chromogenic Western Blot Immunodetection Kit (Invitrogen). Subconfluent HepG2 cells were harvested by trypsinization and lysed with universal lysis/immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM PMSF, 5 μg of leupeptin/ml, 0.5%(v/v) Triton X-100, 0.5% (v/v) Nonidet (P-40). Protein concentration was determined by the Bradford method (Bio-Rad). Western blotting was performed as described above.