Product Class: Kit

PURExpress® In Vitro Protein Synthesis Kit

Catalog #SizeConcentration
E6800S10 reactions
E6800L100 reactions

Description

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
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

  • PURExpress Solution A
  • PURExpress Solution B (Minus RF123)
  • Control (DHFR) template (10 μl)

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
PURExpress Solution A
PURExpress Solution B
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

Storage Temperature

-80°C

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.

Notes

  1. The DHFR control template is now supplied at 125 ng/µl. Use 2 µl for the 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. 
  2. PURExpress DHFR Control Template sequence files: Fasta GenBank
  3. Storage: All kit components should be stored at -80°C.
  4. Add Solution B to Solution A, do not dilute Solution B unbuffered. We recommend a starting amount 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 25 μl reaction.

References

  1. 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.
  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.
  1. When using PURExpress, I was unable to synthesize the control protein?
  2. When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
  3. When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
  4. Detailed FAQs for PURExpress?
  1. Protein Synthesis Reaction using PURExpress (E6800)
  2. Analysis of Synthesized Protein using PURExpress (E6800)
  3. Determination of Protein Synthesis Yield with PURExpress (E6800)
  4. Purification of Synthesized Protein using Reverse His-tag Purification
  5. Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress

Application Notes

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.