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A simple real-time assay for in vitro translation.

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 O(6)-alkylguanine DNA O(6)-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.

SUBMITTER: Capece MC 

PROVIDER: S-EPMC4338355 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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A simple real-time assay for in vitro translation.

Capece Mark C MC   Kornberg Guy L GL   Petrov Alexey A   Puglisi Joseph D JD  

RNA (New York, N.Y.) 20141218 2

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 O(6)-alkylguanine DNA O(6)-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  ...[more]

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