Cell-Free Expression: Non-Radioactive Detection/Applications

The Transcend™ Non-Radioactive Translation Detection Systems allow nonradioactive detection of proteins synthesized using cell free expression. Using these systems, biotinylated lysine residues are incorporated into nascent proteins during translation, This biotinylated lysine is added to the translation reaction as a precharged ε-labeled biotinylated lysine-tRNA complex rather than a free amino acid. After SDS-PAGE and electroblotting, the biotinylated proteins can be visualized by binding either Streptavidin-Alkaline Phosphatase (Streptavidin-AP) or Streptavidin-Horseradish Peroxidase (Streptavidin-HRP), followed either by colorimetric or chemiluminescent detection. Typically, these methods can detect 0.5–5ng of protein within 3–4 hours after gel electrophoresis and can be used for a variety of proteomics related applications. Examples include:

Kumeta, Y. and Ito, M. (1998) Characterization of δ-Guaiene Synthases from Cultured Cells of Aquilaria, Responsible for the Formation of the Sesquiterpenes in Agarwood Plant Physiology 154, 1998–2007 δ-guaiene is one of the compounds found the resinous portions of the Aquilaria plants. A cDNA library was created and screened for clones that expressed guaiene synthases. Three clones were found and further characterized using both cell based and cell free expression. In this case mutations were created and expressed in the TNT system, labeled with Transcend™ tRNA. Relative expression levels, size of expressed protein and related enzymatic activity were determined for each clone.

Vega-Sanchez, M. et al. (2008) SPIN1, a K homology domain protein negatively regulated and ubiquitinated by the E3 ubiquitin ligase SPL11, is involved in flowering time control in rice. Plant Cell 20, 1456–69. SPL11 is one the key proteins involved in the regulation of flowering in plants. A rice cDNA library was screened using the yeast two hybrid system to determine what proteins interact with SPL11. Those experiments indicated that SPL11 interacted with SPIN1. To characterize the interaction, SPIN1 was expressed using the TNT system, labeled with Transcend™ tRNA and used in conjunction with GST-Pulldowns

Naoe, H. et al. (2010) The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation. Mol.Cell Biol. 30, 3994–4005. Cdh1 is one of the coactivators of the anaphase-promoting complex/cyclosome that functions as an E3 ubiquitin ligase for various cell cycle proteins from anaphase to end of the G1 phase of the cell cycle. Other data suggest that Cdh1 is active in processes other than cell cycle such as the regulation of cell shape. By expressing p190 in the TNT system labeling with Transcend™ tRNA and including components required for ubiquitation, it was determined that Cdh1 targeted p190 for degradation.

Marchive, C. et al. (2007) Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants. J. Experimental Botany 58, 1999–2010. In plants many WRKY proteins are involved in the regulation of plant defenses against attack by phytopathogens such as bacteria. It is assumed that this class of transcription factors binds to the W-box , a common promoter element contained in the SAR gene promoters. To determine if this is the case in cultivated grapevines. A full-length VvWRKY1 cDNA was isolated from a Vitis vinifera grape library. In order to test VvWRKY1 protein–W-box DNA interactions, electrophoretic mobility shift assays (EMSAs) were performed. VvWRKY1 was expressed using TNT, labeled with Transcend™ tRNA and incubated with three oligonucleotide probes previously shown to be bound by WRKY proteins in other plant species.

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Gary Kobs

Gary Kobs

Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and Sr. Product Manager for Protein Analysis products. Gary has retired from Promega Corporation.

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