Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions
Open Access
- 15 February 2002
- journal article
- Published by Cold Spring Harbor Laboratory in Genome Research
- Vol. 12 (3) , 487-492
- https://doi.org/10.1101/gr.218802
Abstract
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. [Online supplementary material available at http://www.genome.org.]Keywords
This publication has 40 references indexed in Scilit:
- Global Analysis of Protein Activities Using Proteome ChipsScience, 2001
- Initial sequencing and analysis of the human genomeNature, 2001
- Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBFNature, 2001
- An Ultrasensitive Bacterial Motor Revealed by Monitoring Signaling Proteins in Single CellsScience, 2000
- Simultaneous Binding of Two DNA Duplexes to the NtrC−Enhancer Complex Studied by Two-Color Fluorescence Cross-Correlation SpectroscopyBiochemistry, 2000
- Effect of Electrostatic Interactions on the Binding of Charged Substrate to GroEL Studied by Highly Sensitive Fluorescence Correlation SpectroscopyBiochemical and Biophysical Research Communications, 2000
- Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA MicroarrayScience, 1995
- The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformationBiochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1991
- Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomesCell, 1986
- Amino and carboxy-terminal regions in globular proteinsJournal of Molecular Biology, 1983