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  • PURExpress® Δ (aa, tRNA) Kit

    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.

    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 ).


    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Control (DHFR) template
    PURExpress Solution B
    E. coli tRNA (25 μl)
    Amino Acid Mix (25 μl)
    Solution A minus aa, tRNA

    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

    Notes

    1. For a positive control reaction, use 2 μl of the supplied DHFR control template and 2.5 μl each of the supplied aa and tRNA.
    2. Each kit contains sufficient reagents for 10 x 25 µl reactions.
      The amino acids and tRNA are supplied separately, allowing users to perform a protein synthesis reaction by adding modified amino acids and tRNA mixture to the reaction.
    3. Add Solution B to Solution A, do not dilute Solution B unbuffered. We recommend a starting concentration of 250 ng template DNA per 25 μl reaction. The optimal amount of input DNA can be determined by setting up multiple reactions and titrating the amount of template DNA added to the reaction. Typically, the optimal amount will fall in a range of 25-1000 ng template per μl reaction.
    4. PURExpress DHFR Control Template sequence files: Fasta, GenBank
    5. The DHFR control template is now supplied at 125 ng/μl. Use 2 μl for a positive control reaction. Template DNA, particularly plasmid DNA prepared by mini-prep (e.g. Qiagen) is often the major source of RNase contamination. We strongly recommend adding 20 units Murine RNase Inhibitor  (NEB #M0314) to each reaction.

    References

    1. Asahara, H and Chong. S. (2010). Nuc. Acid. Res. .
    2. 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.
    3. 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.
    4. 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.
    5. 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.
    6. 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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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].

    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 info@neb.com or fill out the Technical Support Form for appropriate document.
    1. How is the Δ aa Δ tRNA Kit E6840S different from the PURExpress E6800S kit?
    2. When using PURExpress, I was unable to synthesize the control protein?
    3. When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
    4. When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
    5. Detailed FAQs for PURExpress?
    1. Protein Synthesis Reaction using PURExpress® ∆ (aa, tRNA) Kit (E6840)
    2. Analysis of Synthesized Protein using PURExpress (E6840)
    3. Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
    4. Determination of Protein Synthesis Yield with PURExpress (E6840)
    5. Purification of Synthesized Protein using Reverse His-tag Purification
    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.