Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Mita, Paolo; Lhakhang, Tenzin; Li, Donghui; Eichinger, Daniel; Fenyo, David; Boeke, Jef
• Is Part Of: Biological procedures online. Volume 18:Issue 1(2016)
• Page Start: 1
• Page End: 14
• Publication Date: 2016-12
• Subjects: Immunoprecipitation -- Immunoblotting -- High-throughput -- HuEV-A vector -- Antibodies -- Screening; Biology -- Methodology -- Periodicals; Biology -- Research -- Periodicals; 570.5
• DOI: 10.1186/s12575-016-0046-x
• Languages: English
• ISSNs: 1480-9222

Abstract

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.