Western Blot of Membrane Proteins

Total protein lysates of membrane-enriched extracts were prepared, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane for western blotting as described previously (Heffeter et al. 2007 (link)). Primary antibodies used are given in Supplementary Table 2. Secondary, horseradish peroxidase-labeled antibodies from Santa Cruz Biotechnology were used in working dilutions of 1:10,000. Since reference proteins were affected by chronic arsenic treatment and could not be used for a reliable normalization, total protein load via Coomassie staining as outlined by (Eaton et al. 2013 (link)) was used instead.

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Weinmuellner R., Kryeziu K., Zbiral B., Tav K., Schoenhacker-Alte B., Groza D., Wimmer L., Schosserer M., Nagelreiter F., Rösinger S., Mildner M., Tschachler E., Grusch M., Grillari J, & Heffeter P. (2017). Long-term exposure of immortalized keratinocytes to arsenic induces EMT, impairs differentiation in organotypic skin models and mimics aspects of human skin derangements. Archives of Toxicology, 92(1), 181-194.

Publication 2017

horseradish peroxidase-labeled antibodies

Antibodies Arsenic Dilutions Horseradish peroxidase Membrane Membrane protein Polyvinylidene difluoride Protein Sds page

Corresponding Organization :

Other organizations : BOKU University, Comprehensive Cancer Center Vienna, Medical University of Vienna

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Variable analysis

independent variables

Chronic arsenic treatment

dependent variables

Protein expression levels (as measured by western blotting)

control variables

Experimental procedures for protein lysate preparation, SDS-PAGE, and western blotting (as described previously in Heffeter et al. 2007)
Primary antibodies (as listed in Supplementary Table 2)
Secondary, horseradish peroxidase-labeled antibodies (used in 1:10,000 dilution)

controls

No positive controls were explicitly mentioned.
Negative control: Total protein load via Coomassie staining (as outlined by Eaton et al. 2013) was used instead of reference proteins, which were affected by chronic arsenic treatment and could not be used for reliable normalization.

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