DescriptionA 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. The relative nuclease-free and protease-free nature of the PURExpress platform 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.
- RF1 (10 μl)
- RF2 (10 μl)
- RF3 (10 μl)
- Control (DHFR) template (10 μl)
- PURExpress Solution A
- PURExpress Solution B (Minus RF123)
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|RF1 (10 μl)|
|RF2 (10 μl)|
|RF3 (10 μl)|
|Control (DHFR) template|
|PURExpress Solution A|
|PURExpress Solution B|
Advantages and Features
- Quickly generate analytical amounts of protein for further characterization
- Confirmation of open reading frames
- Examination of the effects of mutations on ORFs
- Generation of truncated proteins to identify active domains and functional residues
- Introduction of modified, unnatural or labeled amino acids
- Epitope mapping
- Expression of toxic proteins
- Ribosome display
- Translation and/or protein folding studies
- In vitro compartmentalization
Properties and Usage
Materials Required but not Supplied
- General: 37°C incubator
- Labeling: 35S-Methionine (>1000 Ci/mmol recommended, in vitro translation grade)
- TCA Precipitation: TCA solutions (25%, 10%), 1 M NaOH, casamino acids, ethanol, glass fiber filters, vacuum filtration manifold
- SDS-PAGE: Gels and running buffer, gel apparatus, power supply, gel dryer
- Western Blotting: Transfer apparatus, membrane, antibodies and detection reagent
- Purification: Ni-NTA Agarose, Amicon Ultra- 0.5 ml, Ultracel- 100K Membrane Centrifugal Filters
Legal and Disclaimers
LicensesPURExpress® is based on the PURE System Technology originally developed by Dr. Takuya Ueda at the University of Tokyo and commercialized as the PURESYSTEM® by BioComber (Tokyo, Japan).
Licensed from BioComber (Tokyo, Japan) under Patent Nos. 7,118,883; WO2005-105994 and JP2006-340694. For research use only. Commercial use of PURExpress® Δ RF123 Kit requires a license from New England Biolabs, Inc.
Material Safety Datasheets
- For a positive control reaction, use 2 μl of the supplied DHFR control template and 0.5 μl each of the supplied release factors.
- Each kit contains sufficient reagents for 10 x 25 µl reactions.
- The three release factors are supplied separately, allowing users to perform a protein synthesis reaction/ribosome display experiment with/without release factors of their choice.
- Release factors have not been added to solution B. You may still observe translational termination at a reduced level depending on your application and protein template design.
- Asahara, H. and Chong, S. (2010). In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme.. Nuc. Acid. Res. doi:10.1093/nar/gkq377.
- Noto, T., Kurth, H., Kataoka, K., Aronica, L., DeSouza, L., Siu, K., Pearlman, R., Gorovsky, M. and Mochizuki, K. (2010). The tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell. 140, 692-703.
- Tanner, D., Cariello, D., Woolstenhulme, C., Broadbent, M. and Buskirk, A. (2009). Genetic identification of nascent peptides that induce ribosome stalling.. J. Biol. Chem. 284, 34809-34818.
- Talabot-Ayer, D., Lamacchia, C., Gabay, C., and Palmer, G. (2009). Interleukin-33 is biologically active independently of Caspase-1 cleavage.. J. Biol. Chem. 284, 19420-19426.
- Feng, Y. and Cronan, J. E. (2009). A new member of the Eschericia coli fad regulon: transcriptional regulation of fadM (ybaW).. J. Bacteriol. 191, 6320-6328.
- Solaroli, N., Panayiotou, C., Johansson, M., and Karlsson, A. (2009). Identification of two active functional domains of human adenylate kinase 5.. FEBS Lett. 283, 2872-2876.
- How is the ΔRF123 Kit E6850S different from the PURExpress E6800S kit?
- Detailed FAQs for PURExpress?
- When using PURExpress, I was unable to synthesize the control protein?
- When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
- When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
- Analysis of Synthesized Protein using PURExpress (E6850)
- Determination of Protein Synthesis Yield with PURExpress (E6850)
- Protein Synthesis Reaction using PURExpress® ∆ RF123 Kit (E6850)
- Purification of Synthesized Protein using Reverse His-tag Purification
- Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
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.