Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis

Methods Mol Biol. 2017:1575:175-187. doi: 10.1007/978-1-4939-6857-2_10.


We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is easily scalable, adaptable to different cell lines and conditions and has been shown to be reproducible between multiple laboratories.

Keywords: Antibody fragments; Antibody validation; Immunoprecipitation; Mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibody Specificity
  • HEK293 Cells
  • Humans
  • Immunoprecipitation / methods*
  • Mass Spectrometry / methods*
  • Peptide Library
  • Single-Chain Antibodies / immunology*
  • Single-Chain Antibodies / isolation & purification
  • Single-Chain Antibodies / metabolism


  • Peptide Library
  • Single-Chain Antibodies