Product Class: Kit

PURExpress® Δ RF123 Kit

Catalog #SizeConcentration
E6850S10 reactions


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 ).

Kit Components

  • RF1 (10 μl)
  • RF2 (10 μl)
  • RF3 (10 μl)
  • Control (DHFR) template (10 μl)
  • PURExpress Solution A
  • PURExpress Solution B (Minus RF123)

Kit Components

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

Supporting Documents

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
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].
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 or fill out the Technical Support Form for appropriate document.


  1. For a positive control reaction, use 2 μl of the supplied DHFR control template and 0.5 μl each of the supplied release factors.
  2. Each kit contains sufficient reagents for 10 x 25 µl reactions.
  3. 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.
  4. 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.


  1. Hong, S. H., I. Ntai, et al. (2013). Cell-free Protein Synthesis from a Release Factor 1 Deficient Escherichia coli Activates Efficient and Multiple Site-specific Nonstandard Amino Acid Incorporation. ACS Synthetic Biology. 3(6), 398-409. PubMedID: 24328168
  2. Kogure, H., Y. Handa, et al. (2013). Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ. Nucleic Acids Research. 42(5), 3152-63. PubMedID: 24322300
  1. How is the ΔRF123 Kit E6850S different from the PURExpress E6800S kit?
  2. Detailed FAQs for PURExpress?
  3. When using PURExpress, I was unable to synthesize the control protein?
  4. When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
  5. When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
  6. Are there PURExpress citations?
  1. Analysis of Synthesized Protein using PURExpress (E6850)
  2. Determination of Protein Synthesis Yield with PURExpress (E6850)
  3. Protein Synthesis Reaction using PURExpress® ∆ RF123 Kit (E6850)
  4. Purification of Synthesized Protein using Reverse His-tag Purification
  5. Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
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.