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

Doi, Nobuhide; Takashima, Hideaki; Kinjo, Masataka; Sakata, Kyoko; Kawahashi, Yuko; Oishi, Yuko; Oyama, Rieko; Miyamoto-Sato, Etsuko; Sawasaki, Tatsuya; Endo, Yaeta; Yanagawa, Hiroshi
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /03/2002 EN
Relevância na Pesquisa
383.7161%
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.

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

Ying, Bei-Wen; Fourmy, Dominique; Yoshizawa, Satoko
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /11/2007 EN
Relevância na Pesquisa
400.05938%
We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein–RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.