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Journal of Biomolecular Screening
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High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins

Mamatha Sauermann

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Florian Hahne

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Christian Schmidt

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Meher Majety

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Heiko Rosenfelder

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Stephanie Bechtel

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Wolfgang Huber

EMBL-European Bioinformatics Institute, Cambridge, United Kingdom

Annemarie Poustka

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Dorit Arlt

Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany

Stefan Wiemann

Division of Molecular Genome Analysis German Cancer Research Center-DKFZ Im Neuenheimer Feld 580 69120 Heidelberg, Germany, s.wiemann{at}dkfz.de

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. (Journal of Biomolecular Screening 2007:510-520)

Key Words: apoptosis • caspase-3 • genomics • flow cytometry • cell-based assay

This version was published on June 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 4, 510-520 (2007)
DOI: 10.1177/1087057107301271
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