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Anal Biochem. 2019 Dec 1;586:113417. doi: 10.1016/j.ab.2019.113417. Epub 2019 Sep 7.

Differential effects of hydrogen peroxide (H2O2) treatment on epitope recognition in western blotting.

Author information

1
Leibniz Institute on Aging, Fritz Lipmann Institute, 07745, Jena, Germany; Department of Medical Oncology, Sir Run Run Shaw Hospital, Medical School of Zhejiang University, Hangzhou, 310016, China.
2
Leibniz Institute on Aging, Fritz Lipmann Institute, 07745, Jena, Germany. Electronic address: yan.cui@leibniz-fli.de.
3
Leibniz Institute on Aging, Fritz Lipmann Institute, 07745, Jena, Germany; Faculty of Medicine, Friedrich-Schiller-University Jena, 07743, Jena, Germany.

Abstract

Inactivation of horseradish peroxidase by hydrogen peroxide (H2O2) treatment is a convenient alternative to stripping for sequential chemiluminescent western blotting (WB). However, little evidence exists on whether H2O2 treatment affects epitope recognition. Here we show that H2O2 treatment had a negligible effect for most of the tested antibodies, whereas it could also eliminate or enhance antibody binding. Thus, H2O2 treatment has unpredictable effects on epitope recognition. Moreover, we demonstrate potential steric hindrance from previously bound antibodies. Hence, it would be advantageous to pre-test the suitability of antibodies for H2O2-treated WB, and to optimize conditions to mitigate steric interference with re-probing.

KEYWORDS:

Epitope; Erk1/2; H(2)O(2); HRP; Western blotting; YAP

PMID:
31505161
DOI:
10.1016/j.ab.2019.113417

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