4HNE-Protein Adduct Quantification in Cardiac Samples

Western blot was performed as described earlier.^{35 (link),36 (link)} In brief, cardiac protein samples were separated on sodium dodecyl sulphate (SDS)-polyacrylamide gels by electrophoresis and the proteins transferred to immobilon-P membranes (Millipore, Billerica, MA). Levels of 4HNE-protein adducts in heart samples were determined using an antibody of 4HNE-Cys/His/Lys (Calbiochem) at a concentration of 1: 1000. Anti-alpha-tubulin mouse monoclonal antibody at a concentration of 1: 1000 (Santa Cruz Biotechnology, Santa Cruz) was used as a housekeeping marker. The bound antibody was visualized with horseradish peroxidase (HRP)-coupled secondary antibody.

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Mali V.R., Ning R., Chen J., Yang X.P., Xu J, & Palaniyandi S.S. (2014). Impairment of aldehyde dehydrogenase-2 by 4-hydroxy-2-nonenal adduct formation and cardiomyocyte hypertrophy in mice fed a high-fat diet and injected with low-dose streptozotocin. Experimental biology and medicine (Maywood, N.J.), 239(5), 610-618.

Publication 2014

immobilon-P membranes Anti-alpha-tubulin mouse monoclonal antibody

Alpha tubulin Antibody Heart Horseradish peroxidase Immobilon p Membranes Monoclonal antibody Mouse Polyacrylamide gels electrophoresis Protein Sodium dodecyl sulphate Western blot

Corresponding Organization :

Other organizations : Henry Ford Health System, Wayne State University

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Variable analysis

independent variables

Cardiac protein samples

dependent variables

Levels of 4HNE-protein adducts in heart samples

control variables

Anti-alpha-tubulin mouse monoclonal antibody used as a housekeeping marker

controls

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

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