High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins.
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.
Sauermann
Mamatha
M
Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany.
Hahne
Florian
F
Schmidt
Christian
C
Majety
Meher
M
Rosenfelder
Heiko
H
Bechtel
Stephanie
S
Huber
Wolfgang
W
Poustka
Annemarie
A
Arlt
Dorit
D
Wiemann
Stefan
S
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
D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3