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Anti-Nuclear Antibody Screening Using HEp-2 Cells
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Antibody Validation Strategy for Nuclear Receptors

Borbala Katona1, Ahmed Ibrahim2, Mårten Sundberg3

  • 1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

Methods in Molecular Biology (Clifton, N.J.)
|May 2, 2019
PubMed

Antibodies are invaluable biological tools that we can use to detect the presence, location, or alteration of nuclear receptors. However, antibodies frequently cross-react with other proteins and their performance can vary from batch to batch, from application to application and from lab to lab. When each lot of antibody is not thoroughly validated for each assay, each sample type, and each lab and user, antibody-based assays can lead to flawed interpretations and reproducibility problems. In this chapter, we describe a scheme for thorough antibody validation, suitable for nuclear receptors. The method is based on using highly characterized positive and negative controls assembled into a validation tissue microarray (TMA). Through correlation of immunohistochemical staining (IHC) and mRNA levels over multiple tissues, use of current public databases, and assessment of binding to intended and nonintended targets using western blotting (WB), immunoprecipitation (IP), and mass spectrometry (MS), we describe a path for thoroughly validation of antibodies.

Keywords:
AntibodyEstrogen receptorImmunohistochemistryImmunoprecipitationMass spectrometryNuclear receptorsRNA-SeqTissue microarrayValidationWestern blotting

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