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PURExpress® Δ Ribosome Kit
Catalog #
E3313S
Size
10 reactions
Concentration
Categories: Cell-Free Expression
Applications: Cell-Free Protein Expression
Product
Information
FAQs &
Tech Tips
Description
Properties and Usage
Notes
Protocols &
Manuals
Quality &
Safety
Legal
Information
Advantages and Features
Related Products
References
Description
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. 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
PURExpress Citations
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 ).
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 ).
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
Kit Components
The following reagents are supplied with this product:
Store at (°C)
Concentration
Factor Mix
E. coli Ribosome
-80
PURExpress Solution A
Control (DHFR) template
Advantages and Features
Applications
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
Storage Temperature
-80°C
Related Products
Companion Products
PURExpress® Δ (aa, tRNA) Kit
PURExpress® Δ RF123 Kit
PURExpress® In Vitro Protein Synthesis Kit
E. coli Ribosome
PURExpress® Disulfide Bond Enhancer
RNase Inhibitor, Murine
Notes
1. 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.
2. PURExpress Control Template sequence files: Fasta, GenBank
3. Storage: All kit components should be stored at -80°C.
References
1. Gupta, P., K. Kannan, et al. (2013). Regulation of Gene Expression by Macrolide-Induced Ribosomal Frameshifting. Mol Cell. 52(5),
629-42. PubMedID: 24239289
2. 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. PubMedID: 23749080
3. 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. PubMedID: 22741553
4. Kaiser, C. M., D. H. Goldman, et al. (2011). The ribosome modulates nascent protein folding. Science. 334(6063), 1723-7. PubMedID:
22194581
5. Kannan, K., N. Vázquez-Laslop, et al. (2012). Selective Protein Synthesis by Ribosomes with a Drug-Obstructed Exit Tunnel. Cell.
151(3), 508-520. PubMedID: 23101624
6. 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. PubMedID: 24158443
7. 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. PubMedID: 24137004
8. Orelle, C., S. Carlson, et al. (2013). Tools for Characterizing Bacterial Protein Synthesis Inhibitors. Antimicrob Agents Chemother.
57(12), 5994-6004. PubMedID: 24041905
9. Shi, W., X. Zhang, et al. (2011). Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science. 333(6049), 16301632. PubMedID: 21835980
10. 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.
11. 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. PubMedID: 20676057
12. Vázquez-Laslop, N., H. Ramu, et al. (2011). Nascent peptide-mediated ribosome stalling promoted by antibiotics. Ribosomes. 377392.
FAQs
Tech Tips
FAQs
1.
2.
3.
4.
5.
Detailed FAQs for PURExpress?
How is the Δ Ribosome Kit E3313S different from the PURExpress E6800S kit?
When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
When using PURExpress, I was unable to synthesize the control protein?
When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low
yield?
6. Where can find all IMPACT FAQs?
7. Are there PURExpress citations?
Tech Tips
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.
Protocols
Datacards
Manuals
Protocols
1.
2.
3.
4.
5.
Protein Synthesis Reaction using PURExpress (E3313)
Analysis of Synthesized Protein using PURExpress (E3313)
Determination of Protein Synthesis Yield with PURExpress (E3313)
Purification of Synthesized Protein using Reverse His-tag Purification
Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
Manuals
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The
following file naming structure is used to name these document files: manual[Catalog Number].
manualE3313
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and
quality controls. The following file naming structure is used to name the majority of these document files: [Catalog
Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the
Technical Support Form for appropriate document.
E3313Datasheet-Lot0141501
Safety Data Sheet
Datacards
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
Factor Mix (30 μl)
E. coli Ribosome
PURExpress Solution A
Control (DHFR) template (10 μl)
Datacards
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and
quality controls. The following file naming structure is used to name the majority of these document files: [Catalog
Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the
Technical Support Form for appropriate document.
E3313Datasheet-Lot0141501
Legal and Disclaimers
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc
(NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes
in humans or animals.
Licenses
PURExpress® 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® In vitro Protein Synthesis Kit requires a license from New England Biolabs, Inc. This
product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in
humans or animals.