Western Blot Analysis Procedure

Western blot analysis was performed as previously described (Jang et al., 2018 (link)). The cells were lysed in lysis buffer [25 mM Tris (pH 7.5), 250 mM sodium chloride (NaCl), 5 mM EDTA, 1% Nonidet P-40 (NP-40), 100 μg/mL phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma-Aldrich)]. The same amount of protein was denatured by boiling at 100°C for 5 min in the sample buffer (Bio-Rad Laboratories, Hercules, CA, USA). Total proteins were separated using 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were probed with primary antibodies overnight, incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), and then visualized using an enhanced chemiluminescence (ECL) detection system (GE Healthcare Life Sciences).

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Kim M.J., Kang Y.J., Sung B., Jang J.Y., Ahn Y.R., Oh H.J., Choi H., Choi I., Im E., Moon H.R., Chung H.Y, & Kim N.D. (2020). Novel SIRT Inhibitor, MHY2256, Induces Cell Cycle Arrest, Apoptosis, and Autophagic Cell Death in HCT116 Human Colorectal Cancer Cells. Biomolecules & Therapeutics, 28(6), 561-568.

Publication 2020

protease inhibitor cocktail sample buffer horseradish peroxidase-conjugated secondary antibodies ECL) detection system

Antibodies Buffer Cells Chemiluminescence Edta Horseradish peroxidase Membranes Nacl Nonidet p 40 Phenylmethylsulfonyl fluoride Polyvinylidene difluoride Protease inhibitor Proteins Sds page Tris Western blot analysis

Corresponding Organization :

Other organizations : Pusan National University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels

control variables

Lysis buffer composition: 25 mM Tris (pH 7.5), 250 mM sodium chloride (NaCl), 5 mM EDTA, 1% Nonidet P-40 (NP-40), 100 μg/mL phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma-Aldrich)
Denaturation conditions: Boiling at 100°C for 5 min in the sample buffer (Bio-Rad Laboratories, Hercules, CA, USA)
Protein separation: 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Protein transfer: Polyvinylidene difluoride (PVDF) membranes
Primary antibodies: Incubated overnight
Secondary antibodies: Horseradish peroxidase-conjugated (Santa Cruz Biotechnology)
Visualization: Enhanced chemiluminescence (ECL) detection system (GE Healthcare Life Sciences)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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