The TnT® Coupled Reticulocyte Lysate Systems offer researchers an alternative for eukaryotic cell-free protein expression: a single-tube, coupled transcription/translation system. The TnT® Lysate Systems greatly simplify the process and reduce the time required to obtain in vitro translation results. Standard rabbit reticulocyte lysate translations commonly use RNA synthesized in vitro from SP6, T3 or T7 RNA polymerase promoters and require three separate reactions with several steps between each reaction. The TnT® Systems bypass many of these steps by incorporating transcription directly in the translation mix.
For optimal protein expression using the TnT® SP6 RNA polymerase, we recommend titrating magnesium acetate in 0.1mM increments between 0.1mM and 0.5mM. In some instances the addition of 0.2mM magnesium acetate has been shown to increase protein expression by 40%. Magnesium acetate is supplied only with Cat.# L4600 and L4601.
For more information about cell-free protein expression, see the Protein Expression chapter of the Protocols & Applications Guide.
Applications
- GST pull-downs.
- Gel shift assays.
- Co-immunoprecipitation.
- Characterization of protein modifications.
- Protein:RNA interactions.
- Protein activity studies.
- Confirmation of gene products.
- Protein Truncation Test (PTT).
References
- Pelham, H.R.B. and Jackson, R.J. (1976) Eur. J. Biochem. 67, 247–56.
- Krieg, P.A. and Melton, D.A. (1984) Nucl. Acids Res. 12, 7057–70.