PURExpress® delta ribosome kit is a variation of the PURExpress In vitro Protein Synthesis Kit where ribosomes are omitted from the translation mix.
Control ribosomes provided separately
Designed for use with your own ribosomes
User supplied ribosomes can be E. coli-based wild type, mutant or ribosome from other bacterial species
Convenient for directly assaying ribosomal activity and translation studies
Product Information
A rapid method for gene expression analysis, PURExpress® is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation. With minimal nuclease and protease activity, the PURExpress system preserves the integrity of DNA and RNA templates/complexes and results in proteins that are free of modification and degradation. Transcription and translation are carried out in a one-step reaction, and require the mixing of only two tubes. With results available in a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies.
Figure 1: Protein expression using the PURExpress® In Vitro Protein Synthesis Kit
25 μl reactions containing 250 ng template DNA and 20 units RNase Inhibitor were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703, discontinued and replaced with NEB #P7717).
Figure 2: Incorporation of 35S-methionine enables visualizationof protein by autoradiography
25 μl reactions containing 250 ng template DNA, 20 units RNase Inhibitor and 2 μl 35S-met were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE, the gel was fixed for 10 minutes, dried for 2 hours at 80°C and exposed to x-ray film for 5 hours at -80°C.
Figure 3: Schematic diagram of protein synthesis and purification by PURExpress Figure 4: Expression and reverse purification of DHFR (A) and T4 DNA Ligase (B) using PURExpress
125 μl reactions were carried out according to recommendations in the accompanying manual. Samples were analyzed on a 10–20% Tris-glycine gel and stained with Coomassie Blue. Note that in both cases, the desired protein can be visualized in the total protein fraction. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703, discontinued and replaced with NEB #P7717 ).
Highlights
Cleaner System - sample degradation eliminated
Easy-to-use - protein expression complete in approximately two hours
Simple Analysis - protein can often be visualized directly on a Coomassie stained gel
This product is related to the following categories:
The DHFR control template is now supplied at 125 ng/µl. Use 2 µl for the positive control reaction. We use 60 pmoles of ribosomes in a standard 25 μl reaction. The supplied control ribosomes are enough for two reactions. Note: Using a smaller amount of ribosomes is possible but the protein yield may be lower. For detailed usage information please refer to the product manual.
PURExpress Control Template sequence files: Fasta, GenBank
Storage: All kit components should be stored at -80°C.
References
Gupta, P., K. Kannan, et al. (2013). Regulation of Gene Expression by Macrolide-Induced Ribosomal Frameshifting. Mol Cell. 52(5), 629-42.
Tsai, A., J. Chen, et al. (2013). Observing Prokaryotic Translation Elongation in Real-Time using Single-Molecule Fluorescence. Biophysical Journal. 104(2, Supplement 1), 257a.
Vazquez-Laslop, N., H. Ramu, et al. (2010). The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide. EMBO J. 29(18), 3108-3117.
Vázquez-Laslop, N., H. Ramu, et al. (2011). Nascent peptide-mediated ribosome stalling promoted by antibiotics. Ribosomes. 377-392.
Gupta, P., S. Sothiselvam, et al. (2013). Deregulation of translation due to post-transcriptional modification of rRNA explains why erm genes are inducible. Nat Commun . 4, 1984.
Harvey, C. J., J. D. Puglisi, et al. (2012). Precursor directed biosynthesis of an orthogonally functional erythromycin analogue: selectivity in the ribosome macrolide binding pocket. J Am Chem Soc. 134(29), 12259-65.
Kaiser, C. M., D. H. Goldman, et al. (2011). The ribosome modulates nascent protein folding. Science. 334(6063), 1723-7.
Kannan, K., N. Vázquez-Laslop, et al. (2012). Selective Protein Synthesis by Ribosomes with a Drug-Obstructed Exit Tunnel. Cell. 151(3), 508-520.
Kopaskie, K. S., K. G. Ligtenberg, et al. (2013). Translational regulation of Yersinia enterocolitica mRNA encoding a type III secretion substrate. Journal of Biological Chemistry. 288(49), 35478-88.
Martínez, A. K., E. Gordon, et al. (2013). Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan. Nucleic Acids Research. 42(2), 1245-56.
Orelle, C., S. Carlson, et al. (2013). Tools for Characterizing Bacterial Protein Synthesis Inhibitors. Antimicrob Agents Chemother. 57(12), 5994-6004.
Shi, W., X. Zhang, et al. (2011). Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science. 333(6049), 1630-1632.
Thaw and assemble reactions on ice
Thoroughly mix solutions A and B before using. Do not vortex Solution B or ribosomes, mix gently.
Solution A may have a cloudy white appearance. Add to the reaction as a uniform suspension.
Assemble the reactions in the following order on ice: Solution A, Solution B, RNAse Inhibitor, Water, Template DNA or RNA
Once reaction is assembled take time to make sure everything is thoroughly mixed by gently pipetting up and down, pulse spin and place at 37C for 2 to 4 hours.
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Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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