title: Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.
abstract: Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.
authors.affiliation: Microfluidics Systems Biology Lab, Institute of Molecular and Cell Biology (IMCB), A*STAR , 138673, Singapore.
authors.forename: Lih Feng
authors.initials: LF
authors.lastname: Cheow
authors.forename: Ramya
authors.initials: R
authors.lastname: Viswanathan
authors.forename: Chee-Sing
authors.initials: CS
authors.lastname: Chin
authors.forename: Nancy
authors.initials: N
authors.lastname: Jennifer
authors.forename: Robert C
authors.initials: RC
authors.lastname: Jones
authors.forename: Ernesto
authors.initials: E
authors.lastname: Guccione
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Quake
authors.forename: William F
authors.initials: WF
authors.lastname: Burkholder
mesh_terms: D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D057166:High-Throughput Screening Assays / Q000191:economics*; D006657:Histones / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013997:Time Factors
chemical_list: D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D008024:Ligands; D010455:Peptides