笔记详情
标题
Western Blot Analysis
内容
Western Blot Analysis
To obtain total protein lysates, cancer cells or tissues were lysed in 50 mM Tris (pH 7.2), 1% Triton X-100 containing Halt Protease Inhibitor cocktail (Pierce, Rockford, Illinois, United States) and centrifuged at 12,000g for 15 min at 4 °C. To obtain nuclear extracts, NE-PER Nuclear Extraction Reagents (Pierce) were used. Protein concentrations were estimated by the BCA method (Pierce).
For immunoblot analysis, 30 μg and 100 μg of protein from nuclear extracts and total protein lysates, respectively, were used and resolved on 10% SDS-PAGE gels. Proteins were transferred onto PVDF membranes, and the following antibodies were used for immunoblotting: anti-KEAP1 (gift from M. Velichkova, University of California San Diego, La Jolla, California, United States), anti-NRF2 (H-300; Santa Cruz Biotechnology, Santa Cruz, California, United States), anti–Lamin B1 (Santa Cruz Biotechnology), and anti-GAPDH (Imgenex, Sorrento Valley, California, United States). All primary antibodies were diluted in PBS-T/5% nonfat dry milk and incubated overnight at 4 °C.
来源
PLoS Med. 2006 October; 3(10): e420.
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