Apoptosis

, Volume 18, Issue 10, pp 1154–1162

Poor antibody validation is a challenge in biomedical research: a case study for detection of c-FLIP

  • Octavian Bucur
  • Bodvael Pennarun
  • Andreea Lucia Stancu
  • Monica Nadler
  • Maria Sinziana Muraru
  • Thierry Bertomeu
  • Roya Khosravi-Far
Original Paper

Abstract

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

Keywords

c-FLIP Apoptosis Antibodies Methods Technologies 

Abbreviations

TRAIL

TNF-related apoptosis inducing ligand

DISC

Death-inducing signaling complex

TNF

Tumor necrosis factor

c-FLIP

Cellular FLICE-inhibitory protein

MAPK

Mitogen-activated protein kinases

ERK

Extracellular signal-regulated kinase

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Octavian Bucur
    • 1
    • 2
  • Bodvael Pennarun
    • 1
  • Andreea Lucia Stancu
    • 1
  • Monica Nadler
    • 1
  • Maria Sinziana Muraru
    • 1
  • Thierry Bertomeu
    • 1
  • Roya Khosravi-Far
    • 1
  1. 1.Department of PathologyHarvard Medical School and Beth Israel Deaconess Medical CenterBostonUSA
  2. 2.Department of Molecular Cell BiologyInstitute of Biochemistry of the Romanian AcademyBucharestRomania

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