title: Detecting kinase activities from single cell lysate using concentration-enhanced mobility shift assay.
abstract: Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.
authors.affiliation: Department of Electrical Engineering and Computer Science and ‡Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Lih Feng
authors.initials: LF
authors.lastname: Cheow
authors.forename: Aniruddh
authors.initials: A
authors.lastname: Sarkar
authors.forename: Sarah
authors.initials: S
authors.lastname: Kolitz
authors.forename: Douglas
authors.initials: D
authors.lastname: Lauffenburger
authors.forename: Jongyoon
authors.initials: J
authors.lastname: Han
mesh_terms: D000595:Amino Acid Sequence; D056945:Hep G2 Cells; D006801:Humans; D010770:Phosphotransferases / Q000378:metabolism*; D059010:Single-Cell Analysis
chemical_list: D010770:Phosphotransferases