title: Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP.
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.
authors.affiliation: Institute of Systems Genetics (ISG), Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, ACLSW Room 560, 430 East 29th Street, New York, NY 10016 USA ; High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
authors.forename: Paolo
authors.identifier: 0000-0002-2093-4906
authors.initials: P
authors.lastname: Mita
authors.affiliation: Center for Health Informatics and Bioinformatics, and Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, New York, NY USA.
authors.forename: Tenzin
authors.initials: T
authors.lastname: Lhakhang
authors.affiliation: Institute of Systems Genetics (ISG), Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, ACLSW Room 560, 430 East 29th Street, New York, NY 10016 USA ; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA ; High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
authors.forename: Donghui
authors.initials: D
authors.lastname: Li
authors.affiliation: CDI Labs, Inc., Mayaguez, Puerto Rico.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Eichinger
authors.affiliation: Center for Health Informatics and Bioinformatics, and Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, New York, NY USA.
authors.forename: David
authors.initials: D
authors.lastname: Fenyo
authors.affiliation: Institute of Systems Genetics (ISG), Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, ACLSW Room 560, 430 East 29th Street, New York, NY 10016 USA ; High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA.
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
keywords: Antibodies; High-throughput; HuEV-A vector; Immunoblotting; Immunoprecipitation; Screening