Cells or liver tissue was homogenized using lysis buffer with containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). Total protein (30 µg) from each sample was separated on a 10% SDS-polyacrylamide gel, and then transferred onto PVDF membranes. Membranes were incubated for 1 h in blocking buffer containing 5% milk, then incubated with anti-β-actin (Santa Cruz Biotechnology, CA), anti-AR (Abcam, CA), or rabbit anti-TLR4 (PL Laboratories, British Columbia), diluted 1:2000 overnight at 4 °C. After overnight incubation, membranes were incubated with peroxidase conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA) for 1 h at room temperature. Proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) and detected with Alpha Ease FC software (Bio-Rad, Hercules, CA) 22 (link).
Alpha ease fc software
Manufactured by Bio-Rad
Sourced in United States
The Alpha Ease FC software is a digital imaging and analysis platform designed for the acquisition and analysis of chemiluminescent, fluorescent, and colorimetric signals. The software provides tools for image capture, processing, and quantification of data from various gel electrophoresis and blotting techniques.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemiluminescent hrp substrate, by Merck Group (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Anti ar, by Abcam (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Mouse anti stat3, by Cell Signaling Technology (1 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions)
Rabbit anti p stat3 tyr705, by Cell Signaling Technology (1 mentions)
Rabbit anti cox 2, by Abcam (1 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p nf κb p65, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by BestBio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Beyotime (1 mentions)
4 protocols using alpha ease fc software
1
Western Blot Analysis of Protein Markers
2
Protein Expression Analysis via Western Blot
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Tumor cells were lysed in the lysis buffer with a protease and phosphatase inhibitor cocktail. Western blotting was performed as standard protocol. Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) was used for visualization and the signal was analyzed with Alpha Ease FC software (Bio-Rad, Hercules, CA).
3
Western Blot Analysis of Signaling Proteins
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Tumor cells were lysed in lysis buffer with a protease inhibitor cocktail (310003, BestBio). Western blotting was performed as previously described.12 (link) Antibodies were used as below: mouse anti-β-actin (sc-47778, Santa Cruz Biotechnology); rabbit anti-NF-κB p65 (#4764, Cell Signaling Technology); rabbit anti-p-NF-κB p65 (#3033, Cell Signaling Technology); mouse anti-STAT3 (#9139, Cell Signaling Technology); rabbit anti-p-Tyr705-STAT3 (#9145, Cell Signaling Technology); rabbit anti-CyclinD1 (BS1741, Bioworld Technology); rabbit anti-TLR4 (PL0402123) and anti-P-gp (PL0304068, PL Laboratories); rabbit anti-COX-2 (ab102005, Epitomics). Proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) and detected with Alpha Ease FC software (Bio-Rad, Hercules, CA).
4
Protein Expression Analysis in Spinal Tissue
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L4-5 spinal segmental tissue or cultured cells were homogenized for 30 min on ice in RIPA lysis buffer (Beyotime, China) containing a mixture of phosphatase and proteinase inhibitors. The sample was centrifuged at 4 °C at 16,000 rpm for 20 min, and then the protein supernatant was collected. The concentration of total protein was measured by BCA protein assay (Beyotime, China). Equal amounts of total proteins (30 µg/lane) were separated on a 10% SDS-PAGE gel and then transferred onto NC membranes. The membrane was blocked with 5% nonfat milk in PBS at RT for 1 h and incubated with antibodies against GFAP (1:1000, rabbit, Abcam), IBA1 (1:500, rabbit, Abcam), NF‐κB (1:500, rabbit, Abcam) and GAPDH (rabbit, 1:5000; CST) overnight at 4 °C. This membrane was washed and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beyotime) at RT for 1 h. The signals were developed using Pierce ECL western blotting Substrate. The intensity of blots was quantified with densitometry using Alpha EaseFC software (Bio-Rad, USA). All protein bands were normalized to GAPDH.
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