Cell lysates or immunoprecipitates were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose membranes (GE Healthcare Sciences). Membranes were blocked in Tris-buffered saline (TBS, pH 7.4) containing 5% nonfat milk and 0.1% Tween-20, washed twice in TBS containing 0.1% Tween-20, and incubated with primary antibody overnight at 4 °C followed by the secondary antibody for 1 h at room temperature. Proteins of interest were visualized using the Enhanced Chemiluminescence (ECL) system (Santa Cruz Biotechnology). Densitometry analysis of protein bands was performed on Gel-Pro Analyzer software. The resources and other information on antibodies are listed in Supplementary Table 1 .
Enhanced chemiluminescence ecl system
Manufactured by Santa Cruz Biotechnology
The Enhanced Chemiluminescence (ECL) system is a laboratory equipment used for the detection and visualization of proteins in Western blot analysis. It utilizes a luminescent chemical reaction to produce light, which is then captured and measured to quantify the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Mouse anti human gapdh monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
4 protocols using enhanced chemiluminescence ecl system
1
Western Blot Analysis of Protein Targets
2
ACE2 Expression in Cell Lines
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Protein samples (30 µg) obtained from lysis in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) of Caco-2, MDA-MB-231, and HL-mEC cells were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Sigma, Burlington, MA, USA). After being blocked with 3% BSA in tris buffered saline buffer containing 0.05% Tween 20, the blot was probed with mouse anti-human ACE2 monoclonal antibody (1:500 dilution; Santa Cruz Biotechnology; clone E-11) and with mouse anti-human GAPDH monoclonal antibody (1:1000 dilution; Santa Cruz Biotechnology; clone G-9). The antigen–antibody complexes were detected using peroxidase-conjugated goat anti-mouse IgG (Sigma) and revealed using the enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology).
3
Western Blot Analysis of GRWE Effects
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CT26 cells (3×105 cells/well) were seeded in a 6-well plate and treated with GRWE (10, 50 and 100 µg/ml). After treatment for 24 h, the cells were lysed with ice-cold lysis buffer (iNtRON Biotech, Seoul, Korea) for 1 h. Total lysates were centrifuged at 14,000 × g for 10 min, and the supernatants were collected for the determination of the extracted protein concentration using the Lowry method. Samples were mixed with 2X buffer, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 5% skim milk for at least 1 h. After washing with 0.1% PBST (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies for 3 h, followed by their treatment with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein blots were detected using an enhanced chemiluminescence (ECL) system (Santa Cruz Biotechnology, Inc.). The density of western blot bands was quantified by densitometric analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Protein Expression
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Cell lysates or immunoprecipitates were subjected to SDS–PAGE, and proteins were transferred to nitrocellulose membranes (GE Healthcare Sciences). Membranes were blocked in Tris-buffered saline (TBS, pH7.4) containing 5% nonfat milk and 0.1% Tween-20, washed twice in TBS containing 0.1% Tween-20 and incubated with primary antibody overnight at 4°C followed by secondary antibody for 1 h at room temperature. Proteins of interest were visualized using the Enhanced Chemiluminescence (ECL) system (Santa Cruz Biotechnology). WB was performed for 2–3 times from at least two independent experiments and representative pictures were shown.
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