Protein Synthesis Reaction using PURExpress® ∆ RF123 Kit (E6850)
Using a positive control template to verify protein synthesis can be useful when unfamiliar with in vitro transcription-translation protocols. We recommend wearing gloves and using nuclease-free tubes and tips to avoid introducing nucleases to your samples. Please keep all reagents on ice before and during the assembly of reactions and avoid multiple freeze-thaw cycles of the tubes. Reactions are typically 25 μl but can be scaled down or up, as needed. Reactions are usually assembled in nuclease-free 0.5 ml microfuge tubes.
- Thaw the necessary number of aliquots of solution A and B on ice. Pulse-spin in microfuge to collect solutions to bottom of tube.
Certain components in Solution A may precipitate during storage. Be sure to mix it well prior to assembling reactions. The performance of the kit will not be compromised.
- Assemble the reaction on ice in a new tube in the following order:
Solution A: 10 μl
Solution B (minus RF123): 7.5 µl
RF1 (if necessary) : 0.5 µl
RF2 (if necessary): 0.5 µl
RF3 (if necessary): 0.5 μl
Supplements (RNase Inhibitor, 35S-met, etc.): X μl
Nuclease-free H2O: X μl
Template DNA: X μl
Total: 25 μl
Incubate at 37°C for at least 2 hours. Additional incubation time (maximum 4 hours) at 37°C may increase yield.
These formulations allow an increase in the "user added" volume (for template, supplements, etc.); tolerating up to 20% over volume (30 μl reaction total) without an appreciable drop in productivity.
The DHFR control template is 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 RNase Inhibitor, Murine (NEB #M0314 ) in each reaction.
For target proteins requiring disulfide bonding, we suggest supplementing the reactions with the PURExpress Disulfide Bond Enhancer (PDBE, NEB #E6820 ). 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. (NEB #E3313)The standard reaction contains 60 pmoles of ribosomes in a 25 μl reaction. The supplied control ribosomes are enough for two reactions. Using a smaller amount of ribosomes is possible but the protein yield may be lower.
- Mix gently and pulse-spin in microfuge to collect mixture at the bottom of the tube.
- Incubate at 37°C for 2 hour.
We recommend using an incubator rather than a water bath, to prevent evaporation. Some reactions can benefit from an additional hour of incubation to achieve maximum yield. Some proteins are also more soluble at reduced temperatures; however, incubating reactions below 37°C will likely reduce yield.
- Stop the reaction by placing the tube(s) on ice.
- Use samples for analysis or purification or freeze at -20°C for use at a later time.
Some material may precipitate during storage at -20°C. Please ensure everything is resuspended by flicking the reaction tube after thawing.
The PURExpress components are highly purified and present in known quantities. The reconstituted nature of this product makes it amenable to modifications. As such, it is easy to perform in vitro labeling reactions with 35S-methionine to allow visualization of the product. It is also straightforward to supplement the reactions with a component under investigation that is believed to have an effect on transcription or translation. In vitro labeling with 35S-methionine can be performed by setting up a standard reaction with the addition of 2 μl of 35S-methionine.
All amino acids, including methionine, are present at approximately 0.3 mM in PURExpress (with the exception of NEB #E6840 ). Labeled amino acids will compete with existing normal amino acids and the observed signal from the label depends on the efficiency of incorporation into the protein of interest. When supplemented with 1.2 μM 35S- L-methionine, we observe levels of incorporation compatible with autoradiographic detection of the synthesized protein. Reactions (1–5 μl) can then be directly resolved by SDS-PAGE (no need for acetone precipitation), the gels are then briefly fixed in a methanol /acetic acid solution (45%/10%) for 5 minutes at 25°C and dried down onto filter paper (2 hrs at 80°C). The dried gel is then exposed to autoradiographic film (overnight at -20°C) or detected with a phosphorimager.
We encourage safe handling of radioisotopes and suggest consulting with your institution’s radiation safety officer for guidelines and advice on the practical aspects of performing labeling reactions in your workplace.