After the treatment, the medium was aspirated. Cells were rinsed once with ice-cold PBS, harvested, and lysed directly with 1X sodium dodecyl sulfate (SDS) gel-loading buffer (50 mM Tris-HCl, pH 6.8, 2% 2-mercaptoethanol, 0.1% bromophenol blue, and 10% glycerol). Samples were heated at 95°C for 5 min and equal amounts of the whole cell extracts were electrophoresed on 8%-15% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to Immunobilon-P (Millipore, USA). Membranes were blocked with Tris-buffered saline with Tween 20 (TBST) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% non-fat milk for 1 h at room temperature to minimize nonspecific binding. After hybridization with the primary antibodies, membranes were washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. After washing three times with TBST, the protein bands were detected using an ECL detection reagent (PerkinElmer [USA] or Millipore). β-Actin was used as the internal control.
Immunobilon p
Manufactured by Merck Group
Sourced in United States
Immunobilon-P is a laboratory product used for Western blotting applications. It is a type of polyvinylidene fluoride (PVDF) membrane designed for the efficient transfer and immobilization of proteins from electrophoresis gels.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection reagent, by PerkinElmer (1 mentions)
Trans blot semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Lamin β1, by Abcam (1 mentions)
Nrf2 primary antibody, by Proteintech (1 mentions)
α tubulin, by GeneTex (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Gtx115378, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Fujifilm (1 mentions)
Las 2000, by Fujifilm (1 mentions)
4 protocols using immunobilon p
1
Whole-Cell Protein Extraction and Western Blot
2
Western Blot Analysis of chIL-10-V5H6
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Recombinant chIL-10-V5H6 was treated with SDS-PAGE reducing buffer, denatured for 5 min at 100 °C and loaded onto a 4–15% pre-cast Mini-PROTEAN TGX Gels (Bio-Rad) and transferred onto a nitrocellulose membrane (Immunobilon-P, Millipore) using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). After blocking with 0.5% skimmed milk power/PBS solution, the membrane was stained with 1.0 μg/ml of each mAb, followed by incubation with goat anti-mouse IgG1-horseradish peroxidase (Southern Biotec) diluted in 0.5% skimmed milk powder/PBS. Detection was carried out using enhanced chemiluminescence (ECL) (GE Healthcare Life Sciences), according to the manufacturer's instructions.
3
Nrf2 Expression Analysis in HepG2 Cells
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Nuclear and cytoplasmic protein extracts were harvested from HepG2 cells treated with or without PME (100 μg/mL) for 6 h and were prepared as described [12 ]. Protein samples (20 μg) were separated using electrophoresis on a 4%–20% gradient gel prepared by a TOOLS HR Gradient gel solution (TOOLS Biotech, New Taipei City, Taiwan) and transferred to a polyvinylidene difluoride transfer membrane (Immunobilon-P, Millipore, Bedford, MA). Subsequently, the membrane was blocked using 2% bovine serum albumin and then incubated with an Nrf2 primary antibody (1:1000, ProteinTech, Chicago, IL) or lamin β1 (1:1000, Abcam, Cambridge, MA) and α-tubulin (1:1000 GeneTex, Irvine, CA) at 4°C overnight. After incubating with a secondary antibody, the Immobilon™ Western Chemiluminescent HRP Substrate (Merck-Millipore) was used to detect reactive protein signals.
4
Quantifying Autophagy Markers in Cell Lysates
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Cell lysates were prepared with Radio-Immunoprecipitation Assay buffer (RIPA buffer; pH 7.4, 50-mM Tris–HCl, 150 mM NaCl, 1-mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) mixed with 1-mM phenylmethanesulfonyl fluoride (PMSF) as a protease inhibitor. The cell lysates were centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was collected in new tubes. Protein concentration was assayed using the BCA Protein Assay Kit (Wako, #297-73101). The protein was separated by 8–14% SDS-PAGE gel and transferred onto PVDF (Immunobilon-P; Millipore), and the membranes were blocked with 3% skim milk in TBS-T [pH 7.6, 20-mM Tris, 150-mM NaCl, 0.02% polyoxyethylene (20) sorbitan monolaurate] for 1 h at room temperature. After incubation, the membrane was treated with primary antibodies for LC3 (MBL, #PM036), p62 (MBL, #M162-3), NDP52 (GeneTex, #GTX115378), and ß-actin (SantaCruz, #sc-47778) at 1:1,000 dilution, and incubated at 4°C overnight. Afterward, the membrane was washed with TBST and treated with mouse monoclonal (MBL, #330) or rabbit polyclonal (MBL, #458) secondary antibodies (1:2,000) at room temperature for 90 min. The membranes were washed with TBS-T for 30 min and detected by enhanced chemiluminescence (ECL; Cytiva, #RPN2209) using medical X-ray film or a luminescent image analyzer (Fujifilm, LAS-2000).
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