TNT® Coupled Reticulocyte Lysate Systems. A single-tube, coupled transcription/translation system.
The TNTÆCoupled Reticulocyte Lysate Systems(añd)offer researchers analternative for eukaryotic in vitro translations: a single-tube, coupledtranscription/translation system. The TNTÆLysate Systems greatly simplify theprocess and reduce the time required to obtain in vitro translation results(Figure 1). Standard rabbit reticulocyte lysate translations (1) commonly useRNA synthesized in vitro (2) from SP6, T3 or T7 RNA polymerase promotersand require three separate reactions with several steps between each reaction.The TNTÆSystems bypass many of these steps by incorporating transcriptiondirectly in the translation mix.In most cases, the TNTÆLysate reactions produce significantly more protein(two- to sixfold) in a 1.5-hour reaction than standard in vitro rabbit reticulocytelysate translations using RNA templates. In comparisons of 35S incorporation,the TNTÆLysate reactions incorporate significantly more radiolabel than dostandard in vitro translation reactions. Published applications of these systemsinclude:ïTruncation mutation analysis [e.g., the Protein Truncation Test (PTT)]ïDrug screening (affecting translation rates)ïMutation and detection analysis (i.e., enzyme kinetics)ïProtein-protein interactions (using GST fusion proteins)ïImmunoprecipitation of protein complexesïProtein dimerization assaysïLigand-binding region determination/confirmation/competition assaysïProtein structure analysisïElectrophoretic mobility shift assays (EMSAs) for DNA:protein interactionsïDNA footprinting and protein cross-linking studiesïProtein-RNA binding assaysïPost-translational modification testsïIn vitro expression cloning (3; functional genomics)ïVerification/characterization of cloned gene productsFor a complete list of references for these and other applications, please visitour citations database at: www.promega.com/citations/The TNTÆLysate Systems are available in a variety of configurations fortranscription and translation of genes cloned downstream from the SP6, T3 or T7 RNA polymerase promoter. To use these systems, 0.2ñ2.0μg of circular plasmid DNA (or linear DNA for the T3 and T7 Systems, see Note 3,Section III.A) are added directly to TNTÆLysate and incubated in a 50μl reaction for 1.5 hours at 30∞C. Included with the TNTÆLysate Systems are luciferase-encoding control plasmids and Luciferase Assay Reagent(b,d,e),which can be used in a non-radioactive assay for functionally active luciferaseprotein. Starting with circular plasmid DNA, in vitro translation results(autoradiograms) are obtained easily in 8 hours. Alternatively, the Transcendô