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Tytuł:
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Antibody Validation Strategy for Nuclear Receptors.
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Autorzy:
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Katona B; Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Ibrahim A; Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden.
Sundberg M; Olink Proteomics, Uppsala, Sweden.
Williams C; Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden. .; Department of Protein Science, CBH, KTH Royal Institute of Technology, SciLifeLab, Solna, Sweden. .
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Źródło:
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Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2019; Vol. 1966, pp. 79-99.
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Typ publikacji:
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Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
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Język:
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English
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Imprint Name(s):
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Publication: Totowa, NJ : Humana Press
Original Publication: Clifton, N.J. : Humana Press,
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MeSH Terms:
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Validation Studies as Topic*
Antibodies, Monoclonal/*analysis
Blotting, Western/*methods
Immunohistochemistry/*methods
Receptors, Cytoplasmic and Nuclear/*immunology
Gene Expression Profiling ; Humans ; Mass Spectrometry ; Reproducibility of Results ; Tissue Array Analysis/methods
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Grant Information:
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R01 CA172437 United States CA NCI NIH HHS
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Contributed Indexing:
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Keywords: Antibody; Estrogen receptor; Immunohistochemistry; Immunoprecipitation; Mass spectrometry; Nuclear receptors; RNA-Seq; Tissue microarray; Validation; Western blotting
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Substance Nomenclature:
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0 (Antibodies, Monoclonal)
0 (Receptors, Cytoplasmic and Nuclear)
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Entry Date(s):
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Date Created: 20190502 Date Completed: 20190827 Latest Revision: 20190827
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Update Code:
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20240105
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DOI:
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10.1007/978-1-4939-9195-2_7
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PMID:
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31041740
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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.
