Going Green

Researchers routinely prepare proteins in test tubes using the in vitro translation (IVT) reaction.1 These reactions are fast, efficient, and reasonably cost-effective. They are also highly flexible. Proteins can be synthesized either from RNA transcribed in vitro (uncoupled), or from plasmid DNA via a coupled transcription/translation system. In addition, the proteins are easily labeled with [35S]-methionine or biotinylated lysine simply by addition of the labeling reagent to the reaction. Unfo

Written byJeffrey Perkel

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Now a third labeling option is available, which promises to alleviate the shortcomings of other methods. Madison, Wis.-based Promega Corp.'s FluoroTect™ Green_Lys in vitro translation labeling system is a BODIPY^®-FL-labeled lysine residue coupled to tRNA_Lys. BODIPY is a fluorophore, so proteins prepared in vitro may be immediately visualized following electrophoresis, using a laser-based fluorescent imaging system (such as the FluorImager^® SI, FluorImager 595, or Typhoon™ 8600, from Molecular Dynamics, or with the FMBIO^® II from Hitachi Instruments). The BODIPY label has an absorption maximum of 502 nm, and an emission maximum of 510 nm.

To use this system, scientists need only add 1-2 µl of the FluoroTect reagent to a standard IVT reaction. Since it is supplied as a charged tRNA, the labeled tRNALys is immediately available to the ribosomes, without the need for in vitro charging. Because of the ability to detect labeled proteins "in-gel," researchers can avoid traditional ...

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