title: High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins.
abstract: After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.
authors.affiliation: Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany.
authors.forename: Mamatha
authors.initials: M
authors.lastname: Sauermann
authors.forename: Florian
authors.initials: F
authors.lastname: Hahne
authors.forename: Christian
authors.initials: C
authors.lastname: Schmidt
authors.forename: Meher
authors.initials: M
authors.lastname: Majety
authors.forename: Heiko
authors.initials: H
authors.lastname: Rosenfelder
authors.forename: Stephanie
authors.initials: S
authors.lastname: Bechtel
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Huber
authors.forename: Annemarie
authors.initials: A
authors.lastname: Poustka
authors.forename: Dorit
authors.initials: D
authors.lastname: Arlt
authors.forename: Stefan
authors.initials: S
authors.lastname: Wiemann
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000032:analysis* / Q000096:biosynthesis*; D053148:Caspase 3 / Q000032:analysis / Q000096:biosynthesis; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D010865:Pilot Projects
chemical_list: D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3