Protein quantification by chemiluminescent Western blotting: Elimination of the antibody factor by dilution series and calibration curve

doi:10.1016/j.jim.2009.12.007

Journal of Immunological Methods

Volume 353, Issues 1–2, 28 February 2010, Pages 148-150

https://doi.org/10.1016/j.jim.2009.12.007 Get rights and content

Abstract

Quantification of chemiluminescent signals from a Western blot is routinely used to determine the increase or the decrease in protein expression or modification in cell or tissue extracts. However, although scientists readily agree that such a procedure is not quantitative, it is nonetheless used quantitatively in most publications without appropriate controls that would increase the accuracy of the measurement. Here we reexamined this aspect and found that the primary antibody itself influences the relation between the Western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. In that context, we strongly encourage researchers to use dilution series and calibration curve when quantifying protein by Western blot using chemiluminescent signal.

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Acknowledgements

We thank C. Blanc and R.K. Daher for their critical reading of the manuscript. This study was supported by the Canadian Institutes of Health Research Grant MOP-7088 and the Canada Research Chair in Stress Signal Transduction.

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¹: Present address: Héma-Québec, Recherche et développement, 1070 Avenue des sciences de la vie, Québec, QC, Canada G1V 5C3.

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Technical noteProtein quantification by chemiluminescent Western blotting: Elimination of the antibody factor by dilution series and calibration curve

Abstract

Section snippets

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Technical note
Protein quantification by chemiluminescent Western blotting: Elimination of the antibody factor by dilution series and calibration curve