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Updated: May 29, 2026
The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis
Published on: February 5, 2014
Detecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and
Ritika Upadhaya1, Wataru Mizunoya, Judy E Anderson
1Department of Biological Sciences, University of Manitoba, Winnipeg, MB, Canada R3T 2N2.
Abstract:
Western blot detection of multiple proteins is challenged by the need to use antibodies from the same species and the harsh stripping methods that can remove protein or reduce protein antigenicity. Quenching using 27% hydrogen peroxide was developed as an alternative to stripping to inhibit horseradish peroxidase used to detect secondary antibodies. To detect two epitopes with same-species primary antibodies, quenching was followed by incubation in a precomplexed mixture of primary and secondary antibodies for the second epitope plus serum from that species. Both methods will be valuable in specific detection of multiple proteins by Western blotting, and will save time, valuable samples, and reagents.
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