Protocol detail

Western Blot Analysis of Mouse Liver Proteins

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Frozen mouse liver tissue was homogenized in RIPA buffer containing 1x EDTA-free protease inhibitor cocktail (Roche) using Omni Tissue Homogenizer (Omni International). The total protein concentration was determined by Bio-Rad Protein Assay and then equalized to 15 g/L. 25 μg of total protein was used for the western blot assay, performed as previously described (15 (link), 16 (link), 45 (link)). Antibodies used in the western blots are anti-BMAL1 (Cell signaling, #14020), anti-Wee1 (Cell signaling, #4936), and anti-TUBULIN (Sigma-Aldrich, T0198).

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Qu M., Zhang G., Qu H., Vu A., Wu R., Tsukamoto H., Jia Z., Huang W., Lenz H.J., Rich J.N, & Kay S.A. (2023). Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle. Proceedings of the National Academy of Sciences of the United States of America, 120(2), e2214829120.

Publication 2023

Omni Tissue Homogenizer Protein Assay anti-BMAL1 anti-Wee1 anti-TUBULIN

Antibodies Assay Buffer Edta Frozen Liver Mouse Protease inhibitor Protein Rad protein Ripa Tissue Tubulin Western blots

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Variable analysis

independent variables

Homogenization of frozen mouse liver tissue in RIPA buffer containing 1x EDTA-free protease inhibitor cocktail (Roche) using Omni Tissue Homogenizer (Omni International)

dependent variables

Expression levels of BMAL1, Wee1, and TUBULIN proteins measured using western blot assay

control variables

Total protein concentration equalized to 15 g/L
25 μg of total protein used for each western blot assay

controls

Positive control: Not specified
Negative control: Not specified

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