Detecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide

doi:10.1016/j.ab.2011.08.014

Analytical Biochemistry

Volume 419, Issue 2, 15 December 2011, Pages 342-344

https://doi.org/10.1016/j.ab.2011.08.014 Get rights and content

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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Acknowledgments

The authors acknowledge funding by the Canadian Space Agency (9F007-052237/001/SR) and the Natural Sciences and Engineering Research Foundation, a postdoctoral fellowship from Foreign Affairs and International Trade Canada (W.M.), and assistance from W. Snow and S. Lakhi.

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¹: These authors contributed equally to the work.

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Notes & TipsDetecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide

Abstract

Section snippets

Acknowledgments

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Biochim. Biophys. Acta

Biomaterials

Biosens. Bioelectron.

Western immunoblot analysis

Methods Mol. Biol.

Western blotting

Methods Mol. Biol.

Notes & Tips
Detecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide