A simple real-time assay for in vitro translation |
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| Authors: | Mark C Capece Guy L Kornberg Alexey Petrov Joseph D Puglisi |
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| Institution: | 1.Department of Chemistry, Stanford University, Stanford, California 94305, USA;2.Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA |
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| Abstract: | A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O6-alkylguanine DNA O6-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC50) of three common macrolide antibiotics. |
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| Keywords: | SNAP in vitro translation assay single-molecule fluorescence IC50 ribosome activity translation rate |
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