Western Blot Analysis of ALDH2 in Hepatic Cells

Hepatic whole-cell lysates were prepared as described previuosly (49 (link)). The denatured protein samples (20 μg) were fractionated on Any kD Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Hercules, CA). Primary antibodies and the titers used for western blotting were as follows: goat polyclonal anti-ALDH2 (N-14) (sc-48838; Santa Cruz Biotechnology, Santa Cruz, CA;1:1000); rabbit monoclonal anti-β-actin (13E5; Cell Signaling Technology; 1:2000). These were then reacted with the appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Little Chalfont, UK). The signal was visualized using an Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Merck Millipore, Darmstadt, Germany) and was exposed using a ChemiDoc XRS system equipped with Image Lab software (Bio-Rad Laboratories).

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Hirohashi K., Ohashi S., Amanuma Y., Nakai Y., Ida T., Baba K., Mitani Y., Mizumoto A., Yamamoto Y., Kikuchi O., Matsubara J., Yamada A., Miyamoto S., Seno H., Matsuda T, & Muto M. (2019). Protective effects of Alda-1, an ALDH2 activator, on alcohol-derived DNA damage in the esophagus of human ALDH2*2 (Glu504Lys) knock-in mice. Carcinogenesis, 41(2), 194-202.

Publication 2019

Any kD Mini-PROTEAN TGX Precast Gels anti-β-actin horseradish peroxidase-conjugated secondary antibody Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate ChemiDoc XRS system Image Lab software

Actin Antibodies Antibody Gels Goat Hepatic cell Horseradish peroxidase Immobilon Protein Rabbit

Corresponding Organization : Kyoto University

Other organizations : Dana-Farber Cancer Institute

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Protein expression levels of ALDH2 and β-actin

control variables

Protein sample concentration (20 μg)
Antibody titers (anti-ALDH2 1:1000, anti-β-actin 1:2000)
Secondary antibody (horseradish peroxidase-conjugated)

controls

Positive control: Not specified
Negative control: Not specified

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