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Related Experiment Video

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Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells
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Antibody Validation for Estrogen Receptor Beta.

Madeleine Birgersson1, Borbala Katona2, Cecilia Lindskog2

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

Methods in Molecular Biology (Clifton, N.J.)
|February 4, 2022
PubMed
Summary

Thorough validation of estrogen receptor beta (ERβ) antibodies is crucial. Our study found only one specific antibody, revealing ERβ expression in limited tissues, contrary to much published research.

Keywords:
AntibodyEstrogen receptor betaImmunohistochemistryRNA-SeqValidationWestern blotting

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Antibody cross-reactivity can lead to misinterpretations in research.
  • Previous evaluations of anti-estrogen receptor beta (ERβ) antibodies showed limited specificity.
  • Discrepancies exist between reported ERβ protein expression and mRNA evidence in scientific literature.

Purpose of the Study:

  • To present a comprehensive validation scheme for ERβ antibodies.
  • To guide the selection of appropriate positive and negative controls for antibody validation.
  • To address the issue of unreliable ERβ antibody data in published research.

Main Methods:

  • Evaluated 13 anti-ERβ antibodies for specificity using immunohistochemistry.
  • Assessed ERβ protein expression in normal human tissues and cancers.
  • Correlated protein expression with available mRNA evidence and public databases.
  • Described validation steps including on- and off-target binding assessment.
  • Included optional immunoprecipitation and mass spectrometry for corroboration.

Main Results:

  • Only one out of 13 anti-ERβ antibodies demonstrated specificity.
  • Specific antibody revealed ERβ expression in a limited subset of human tissues.
  • Findings align with mRNA expression data but contradict numerous published studies.
  • Significant discrepancies noted regarding ERβ protein expression in various tissues.

Conclusions:

  • Robust antibody validation is essential to ensure reliable experimental outcomes.
  • A standardized validation protocol can improve the accuracy of ERβ research.
  • The presented validation scheme is applicable to immunohistochemistry and Western blotting.
  • Further research is needed to reconcile discrepancies in ERβ expression data.