PURExpress® Δ RF123 kit is a variation of the PURExpress In vitro Protein Synthesis Kit where the release factors 1, 2 and 3 are omitted from the translation mix.
RF1, RF2 and RF3 are supplied separately to allow users to control protein synthesis with/without release factors of their choice
Essentially RF1 and RF3 free, RF2 is greatly reduced to minimal levels
Allows for robust recovery of activity-encoding nucleic acid template in library screening applications
Applications include non-natural amino acid incorporation, ribosome display and mRNA display
Product Information
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. With minimal nuclease and protease activity, the PURExpress system 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, discontinued and replaced with NEB #P7717 ).
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, discontinued and replaced with NEB #P7717 ).
This product is related to the following categories:
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.
PURExpress DHFR Control Template sequence files: Fasta, GenBank
References
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.
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.
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.
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Specifications & Change Notifications
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