Related Experiment Video
Updated: Jan 19, 2026
Western Blotting: Western Transfer, Antibody Detection, and Image Analysis
Published on: April 30, 2023
Differential effects of hydrogen peroxide (H2O2) treatment on epitope recognition in western blotting
Shuting Han1, Yan Cui2, Dario-Lucas Helbing3
1Leibniz 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.
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.
Related Concept Videos
The Western Blot
Western Blotting
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
07:45Western Blotting: Sample Preparation to Detection
09:24Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins
11:43The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis
03:35Chemiluminescent Western Blot Assay for Protein Processing

