Fluorescence ImmunoPrecipitation (FLIP): A Novel Assay for High-Throughput IP

Paolo Mita, Tenzin Lhakhang, Donghui Li, Daniel J. Eichinger, David Fenyo, Jef D. Boeke

Research output: Contribution to journalArticle

Abstract

Background: The immunoprecipitation (IP) assay is a valuable molecular biology tool applied across a breadth of fields. The standard assay couples IP to immunoblotting (IP/IB), a procedure severely limited as it is not easily scaled for high-throughput analysis. Results: Here we describe and characterize a new methodology for fast and reliable evaluation of an immunoprecipitation reaction. FLIP (FLuorescence IP) relies on the expression of the target protein as a chromophore-tagged protein and couples IP with the measurement of fluorescent signal coating agarose beads. We show here that FLIP displays similar sensitivity to the standard IP/IB procedure but is amenable to high-throughput analysis. We applied FLIP to the screening of mouse monoclonal antibodies of unknown behavior in IP procedures. The parallel analysis of the considered antibodies using FLIP and IP/western shows good correlation between the two procedures. We also show application of FLIP using unpurified antibodies (hybridoma supernatant) and we developed a publicly available tool for the easy analysis and quantification of FLIP signals. Conclusions: Altogether, our characterizations of this new methodology show that FLIP is an appealing and reliable tool for any application of high-throughput IP.

Original languageEnglish (US)
Article number16
JournalBiological Procedures Online
Volume18
Issue number1
DOIs
StatePublished - Aug 15 2016

Keywords

  • Antibodies
  • High-throughput
  • HuEV-A vector
  • Immunoblotting
  • Immunoprecipitation
  • Screening

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Mita, P., Lhakhang, T., Li, D., Eichinger, D. J., Fenyo, D., & Boeke, J. D. (2016). Fluorescence ImmunoPrecipitation (FLIP): A Novel Assay for High-Throughput IP. Biological Procedures Online, 18(1), [16]. https://doi.org/10.1186/s12575-016-0046-x