Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins

J Proteome Res. 2017 Jan 6;16(1):147-155. doi: 10.1021/acs.jproteome.6b00821. Epub 2016 Oct 26.


Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Keywords: Cell Atlas; GFP; Human Protein Atlas; antibody validation; confocal microscopy; immunofluorescence; spatial proteomics; subcellular localization.

Publication types

  • Validation Study

MeSH terms

  • Analysis of Variance
  • Antibodies / analysis*
  • Antibodies / chemistry
  • Atlases as Topic
  • Cell Line
  • Fluorescent Antibody Technique / standards*
  • Gene Expression
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Microscopy, Confocal / standards*
  • Reference Standards
  • Reproducibility of Results
  • Staining and Labeling / methods*


  • Antibodies
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins