Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions

AUTOR(ES)

Doi, Nobuhide

FONTE

Cold Spring Harbor Laboratory Press

RESUMO

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein–protein and protein–DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

Documentos Relacionados

• Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY
• High-Throughput Growth Assay for Toxoplasma gondii Using Yellow Fluorescent Protein
• High-throughput protein analysis integrating bioinformatics and experimental assays
• Confocal fluorescence coincidence analysis: An approach to ultra high-throughput screening
• Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase