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 journalArticlepeer-review

    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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