The serum of all the studied groups was lysed in NP-40 lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40). Proteins were separated from the whole cell lysate in a 12% SDS-PAGE. Then, the electrophoresed proteins were blotted onto Amersham™ Hybond® P Western blotting polyvinylidene fluoride membranes (GE10600021 Sigma, Sigma-Aldrich, MO, USA), and incubated with the primary antibodies; rabbit anti-GPC3 (1:500) at 4 °C overnight, and mouse anti-β-actin (1:1,000) (Santa Cruz Biotechnology, Santa Cruz, California, USA) at room temperature for 1 h. Incubation with sheep anti-mouse IgG- horseradish peroxidase (Amersham Pharmacia, United Kingdom) then followed. Quantification of the Western blot bands was performed by image analysis using the ChemiDoc MP imaging system (version 3) made by Bio-Rad (Hercules, California, USA). Relative density of each band was evaluated and normalized with β-actin.
Chemidoc mp imaging system version 3
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc MP imaging system (version 3) is a compact and versatile imaging system designed for gel and blot analysis. It utilizes a high-resolution camera and advanced optics to capture high-quality images of chemiluminescent, fluorescent, and colorimetric samples. The system is capable of imaging a variety of sample types, including Western blots, nucleic acid gels, and protein gels.
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Lab products found in correlation
4 protocols using chemidoc mp imaging system version 3
1
Western Blot Quantification of GPC3 Expression
2
Quantitative Western Blot Analysis
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Tissues lysis was carried out in RIPA buffer followed by separation through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The tissue lysates obtained were mixed with lamellae buffer and boiled for 5 min. This was followed by protein separation through SDS-PAGE and subsequent transfer to Immobilon membrane (Millipore). The antibodies used were anti-Phospho-ERK antibody (Boster Biological Technology, Pleasanton CA, USA, Catalog # P00030) and Smad1/2 antibody (Santa Cruz Biotechnology, Inc. Catalog # sc-7960). Incubation was done in 5% nonfat dry milk, Tris-HCL, 0.1% Tween 20 for 1 hr. This was followed by addition of primary antibodies to the membrane carrying samples and subsequent incubation at 4℃overnight. A 2-hour incubation was carried out at room temperature for secondary antibodies. The samples are washed twice in n 1 × TBS-T. Quantification of the target proteins was done by the densitometric analysis of the immunoblots against the control sample by β-actin protein normalization using Image analysis software on the ChemiDoc MP imaging system (version 3) produced by Bio-Rad (Hercules, CA).
3
Quantifying Exosomal CD81 Protein
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The antibody used was antigen affinity-purified polyclonal sheep IgG anti-rabbit CD81 (Catalog no. 0349509; BioLegend, San Diego, California, USA). Protein was isolated from isolated exosomes using radioimmunoprecipitation assay buffer. Twenty nanograms of protein were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4–20% polyacrylamide gradient gels. Following incubation in 5% nonfat dry milk, Tris hydrochloride, 0.1% Tween 20 for 1 h, CD81 polyclonal–monoclonal antibody was added to one of the membranes including specimen samples and incubated at 4°C overnight. Appropriate secondary antibody was incubated for 2 h at room temperature. After being washed twice n 1 × TBS-T, densitometric analysis of the immunoblots was performed to quantify the amounts of CD81 against housekeeping protein β-actin by image analysis software on the ChemiDoc MP imaging system (version 3) produced by Bio-Rad.
4
Quantitative Immunoblot Analysis of Cleaved Caspase-3
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Cells were washed, lysed, and proteins were isolated. They were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis then transferred to an Immobilon membrane (Millipore) [21 ].
After incubation in 5% non-fat dry milk, Tris-HCL, and 0.1 % Tween 20 for 1 -h, the antigen affinity-purified polyclonal sheep IgG anti-human active cleaved caspase-3 antibody was added to one of the membranes containing specimen samples and incubated at 4 °C overnight. Appropriate secondary antibodies were incubated for 2 h at room temperature. The density-metric analysis of the immunoblots was performed by total protein normalization image analysis software on the ChemiDoc MP imaging system (version 3) produced by Bio-Rad (Hercules, CA).
After incubation in 5% non-fat dry milk, Tris-HCL, and 0.1 % Tween 20 for 1 -h, the antigen affinity-purified polyclonal sheep IgG anti-human active cleaved caspase-3 antibody was added to one of the membranes containing specimen samples and incubated at 4 °C overnight. Appropriate secondary antibodies were incubated for 2 h at room temperature. The density-metric analysis of the immunoblots was performed by total protein normalization image analysis software on the ChemiDoc MP imaging system (version 3) produced by Bio-Rad (Hercules, CA).
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