Protein translocation assays: key tools for accessing new biological information with high-throughput microscopy.
Redistribution technology is a cell-based assay technology that uses protein translocation as the primary readout for the activity of cellular signaling pathways and other intracellular events. Protein targets are labeled with the green fluorescent protein, and stably transfected cell lines are generated. The assays are read using a high-throughput, optical microscope-based instrument, several of which have become available commercially. Protein translocation assays can be formatted as agonist assays, in which compounds are tested for their ability to promote protein translocation, or as antagonist assays, in which compounds are tested for their ability to inhibit protein translocation caused by a known agonist. Protein translocation assays are high-content, high-throughput assays primarily used for profiling of lead series, primary screening of compound libraries, and as readouts for gene-silencing studies using siRNAs. This chapter describes two novel high-content Redistribution assay technologies: (1) The p53:hdm2 GRIP interaction assay, in which one high-content image feature is used for detection of primary hits, whereas a different feature is used to deselect compounds with unwanted mode of action, and (2) application of siRNAs to Redistribution assays, exemplified by knockdown of Akt isoforms in a FKHR translocation assay reporting on the PI3 kinase signaling pathway.
Heydorn
Arne
A
Fisher BioImage ApS, Søborg, Denmark.
Lundholt
Betina K
BK
Praestegaard
Morten
M
Pagliaro
Len
L
D000818:Animals; D001671:Biochemistry / Q000295:instrumentation* / Q000379:methods*; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt