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Antibody Validation for Estrogen Receptor Beta

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Estrogen Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2418))

Abstract

Antibodies can cross-react with proteins other than their intended targets, and antibody-based applications can, if not properly validated, lead to flawed interpretations. When evaluating 13 anti-estrogen receptor beta (ERβ) antibodies in 2017, we concluded that only one of them was specific. Applying this antibody in immunohistochemistry of over 44 different normal human tissues and 20 types of cancers revealed ERβ expression in only a few selected tissues. This aligned with mRNA evidence but contradicted a large set of published literature. ERβ protein expression continues to be reported in tissues without clear support by mRNA expression. In this chapter, we describe how ERβ antibodies can be thoroughly validated and discuss selection of well-characterized positive and negative controls. The validation scheme presented is applicable for immunohistochemistry and Western blotting. The protocol includes evaluation of mRNA evidence, use of public databases, assessment of on- and off-target binding, and an optional step for corroboration with immunoprecipitation and mass spectrometry.

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Acknowledgments

We would like to thank Sandra Andersson, Anna Asplund, Mårten Sundberg, and Margareta Ramström (all previously at Uppsala University) for the original set up of key parts of this method. Data generated by the TCGA Research Network (http://cancergenome.nih.gov/), GTEx (http://www.gtexportal.org/), and the Human Protein Atlas (www.proteinatlas.org) have been utilized. This work was supported by the Swedish Cancer Society (CAN 2018/596) and the Swedish Research Council (2017-01658), and benefitted from funding for the Human Protein Atlas Funding provided by the Knut and Alice Wallenberg Foundation.

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Author notes

  1. Madeleine Birgersson

    Present address: SciLifeLab, Department of Protein Science, KTH—Royal Institute of Technology, Solna, Sweden

Authors and Affiliations

  1. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden

    Madeleine Birgersson & Cecilia Williams

  2. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

    Borbala Katona, Cecilia Lindskog & Fredrik Pontén

  3. SciLifeLab, Department of Protein Science, KTH—Royal Institute of Technology, Solna, Sweden

    Cecilia Williams

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  1. Madeleine Birgersson
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  2. Borbala Katona
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  3. Cecilia Lindskog
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  4. Fredrik Pontén
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  5. Cecilia Williams
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Corresponding author

Correspondence to Cecilia Williams .

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Editors and Affiliations

  1. Sanford School of Medicine, University of South Dakota, Vermillion, SD, USA

    Kathleen M. Eyster

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Birgersson, M., Katona, B., Lindskog, C., Pontén, F., Williams, C. (2022). Antibody Validation for Estrogen Receptor Beta. In: Eyster, K.M. (eds) Estrogen Receptors. Methods in Molecular Biology, vol 2418. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1920-9_1

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  • DOI: https://doi.org/10.1007/978-1-0716-1920-9_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1919-3

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