Total protein was extracted from tissues to detect the levels of caspase-3, 19S proteasome, 20S proteasome and ubiquitin ligase E3 in target tissue via western blotting. The proteins were separated via 12% SDS-PAGE, blotted and probed with rabbit anti-caspase-3 (1:500; Sigma-Aldrich; Merck KGaA; cat. no. C8487), rabbit anti-19S proteasome (1:1,000; Enzo Life Sciences; cat. no. Q9SEI2), rabbit anti-20S proteasome (1:1,000; Abcam; cat. no. ab22673) and rabbit anti-enzyme E3 (1:1,000; Abcam; cat. no. ab84067). A Bradford assay (Bio-Rad Laboratories, Inc.) was used to quantify protein concentrations. The blots were visualized using a chemiluminescence system (Amersham Bioscience; GE Healthcare).
Amersham bioscience
Manufactured by GE Healthcare
Amersham Bioscience is a line of lab equipment manufactured by GE Healthcare. The core function of this product is to provide instrumentation and consumables for life science research, including protein analysis, molecular biology, and cell biology applications.
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Lab products found in correlation
Rabbit anti caspase 3, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Anti p21 mouse monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Hybond ecl nitrocellulose membrane, by Cytiva (1 mentions)
Anti β actin mouse monoclonal antibody, by Cell Signaling Technology (1 mentions)
Hybond, by Cytiva (1 mentions)
2 protocols using amersham bioscience
1
Proteasome and Apoptosis Regulation in Tissues
2
Western Blot Analysis of Signaling Proteins
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Cells were lysed, and equal amounts of proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were transferred onto Hybond™-ECL nitrocellulose membranes (Amersham Biosciences; GE Healthcare) to detect specific proteins. The membranes were then incubated overnight at 4 °C with anti-phospho-AKT1/2/3 rabbit polyclonal antibody (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-ERK mouse monoclonal antibody (1:1000, Santa Cruz Biotechnology), anti-P21 mouse monoclonal antibody (1:1000, Santa Cruz Biotechnology), and anti-β-actin mouse monoclonal antibody (1:1000, Cell Signaling Technologies, Beverley, MA, USA). Labeled proteins were detected using a chemiluminescence detector (Amersham Bioscience; GE Healthcare).
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