[First choice for cell-free protein synthesis]
PUREfrex 2.0® mini is an entry model designed for those who are trying protein synthesis for the first time with a cell-free system.
Recommended for those who have the following requests:
- I have never used a “cell-free systems”, so I want an affordable kit for beginners.
- I have never used cell-free systems, so I would like some support.
- I would like to test in a small volume (10 µL～) whether the target protein can be synthesized.
- I have never used a reconstituted system (PURE system), so I would like to try it.
Ordering through the online e-commerce system is available only to customers in the USA and Canada.
International sales of GeneFrontier's products are carried out by Cosmo Bio USA.
To request a quote or place an order, please contact Cosmo Bio USA.
CONTENTS
Overview
[First choice for cell-free protein synthesis]
PUREfrex 2.0® mini is an entry model designed for those who are trying protein synthesis for the first time with a cell-free system.
Anyone can easily synthesize proteins by simply mixing template DNA and reaction solution.
It is the same product as PUREfrex® 2.0, but by making it smaller in volume, we have achieved an amazingly low price, making it easy to introduce for trial use. PUREfrex® 2.0 mini can perform reactions in as little as 10 µL, allowing you to efficiently obtain results even in small volumes. Furthermore, as a different feature from PUREfrex® 2.0, it also comes with “T7Pro-SD primer” necessary for preparing template DNA using PCR and “RNase-free water” used for preparing the reaction solution. T7Pro-SD primer is common primer to all proteins, including the essential T7 promoter and SD sequence in the 5’UTR of template DNA and is optimized for PUREfrex®.
- Synthesis amount of PUREfrex® 2.0 mini >>> See PUREfrex® 2.0
- Contamination test
- References
GeneFrontier offers the following free support for those new to cell-free systems:
1) Detailed manual for PUREfrex 2.0 mini & email inquiry service
GeneFrontier is currently releasing a detailed manual for PUREfrex® 2.0 mini.
The manual contains all the information you need to use PUREfrex® 2.0 mini, including how to design and prepare template DNA, how to prepare PUREfrex® reaction solution, and examples of protein synthesis experiments.
We also provide email-based support for inquiries for those who cannot understand the information from the manual alone.
- How can I synthesize proteins of human origin?
- What should I do if I cannot see the target protein band?
- What should I do if the target protein has no activity? ...etc.
Please feel free to contact us so that our researchers can answer your questions.
Please see our FAQ for answers to frequently asked questions.
2) DNA Design for PUREfrex®
GeneFrontier offers a free service, "DNA Design for PUREfrex®", to optimize DNA template design for protein synthesis using PUREfrex®.
The design of template DNA is a factor that has an extremely large effect on the amount of target protein synthesized. As shown in the figure below, even with the same amino acid sequence, variations in DNA sequences can lead to different levels of protein production. This effect is independent of biological distinctions such as prokaryotic or eukaryotic. Certain DNA sequences tend to produce higher or lower yields of protein, and these trends have become increasingly apparent.
Figure 1. Effects of codons near N-terminal region on protein yield
A. Codons of N-terminal region of heavy chain (HC) of trastuzumab (Herceptin)
B. Comparison of yields from template DNA with different N-terminal codons
Considering these critical factors, DNA template design can often be a challenging and complex task (also see: 6 Tips about the template DNA Design).
Therefore, GeneFrontier offers a free template DNA design optimization service for those who use PUREfrex® kits. We will optimize the sequence of your target protein using template DNA sequence optimization software [PUREcodeTM].
<Consultation is free of charge!>
Send us the amino acid sequence of your target protein by the link below, and we will return a suitable DNA sequence based on our algorism considering important points for protein expression using PUREfrex®, such as codon optimization for whole ORF and N-terminus, UTRs, etc.
Experiment Workflow
General procedures for using PUREfrex® to synthesize functional proteins are shown in Figure.
Start by preparing the template DNA suitable to the protein synthesis with PUREfrex® and examine whether the protein of interest is synthesized. Redesign the nucleotide sequence of the template DNA if too little protein is synthesized. Add supplement such as molecular chaperones or changing the synthesis conditions if the solubility or activity of the synthesized protein is low or absent. The kit contains enough reagents for examining whether the target protein can be synthesized.
Protocols
The amounts given are for 10 µL reaction. For scaling up the reaction, adjust the volume of reagents accordingly.
1. Thaw Solution I by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
2. Thaw Solution II and III on ice.
3. Mix Solution I, II and III respectively by vortex and centrifuge briefly to collect each solution at the bottom.
4. Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 - 3 ng/µL per 1 kb.
| Water | 3.5-X μL |
| Solution I | 5 μL |
| Solution II | 0.5 μL |
| Solution III | 1 μL |
| Template DNA *1 | X μL |
| Total | 10 μL |
|---|
5. Incubate the tube at 37 °C for 4 - 6 hours on a heat block or a water bath.
6. Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.
- Please visit the template DNA preparation site.
Video - How to use PUREfrex -
Specifications
Kit components <PUREfrex® 2.0 mini>
| Reagent | Quantity for 100 μL rxn |
Description | Storage *1 |
|---|---|---|---|
| Solution Ⅰ |
50 µL | Amino acids, NTPs, tRNAs and substrates for enzymes etc. |
-20 °C |
| Solution Ⅱ |
5 µL | Proteins in 30% glycerol buffer |
-20 °C or -80 °C *2 |
| Solution Ⅲ |
10 µL | Ribosome (20 µM) |
-80 °C *2 |
| DHFR DNA *3 |
10 µL | PCR product containing a gene encoding dihydrofolate reductase (DHFR) from E.coli as a positive control. (20 ng/µL) |
-20 °C |
| T7PRO-SD primer | 5 µL | Primer common to all proteins, including the essential T7 promoter and SD sequence in the 5’UTR of template DNA (10 µM) | -20 °C |
| RNase-Free Water | 500 µL | RNase-Free Water | -20 °C |
- Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening.
- The rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol, and be stored at -80 °C. Divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
- For synthesizing DHFR, add 1 µL of DHFR DNA to 20 µL of reaction. Sequence of DHFR DNA is here.
Datasheet
PF201-0.1-EX
DownloadsSDS
US
DownloadsEU
DownloadsNote
PUREfrex® is developed for in vitro research only. PUREfrex® should not be used for the therapy, diagnostic or administration to animals including human and should not be used as food or cosmetics etc. To avoid the contamination of nuclease, nuclease-free-treated water, reagents and materials should be used. We also recommend wearing gloves and mask.
"PUREfrex® is Registered in U.S. Patent and Trademark Office"
Applications
On the Resources page, you can find posters and technical notes about PUREfrex®.
Additionally, there are papers that use PUREfrex® or PURE system technology.
Related Products
When your protein needs an assistance of molecular chaperone or a formation of a disulfide bond to form a correct conformation, we have supplemental reagents by simply adding to PUREfrex® 2.0 mini.
Cell-free Protein Synthesis Kits
PUREfrex®1.0
for Basic Research and Customized kit
Catalog No.
- PF001-0.25-EX, PF001-2ML-EX, PF001-10ML-EX, PF001-50ML-EX
PUREfrex®2.1
for Forming Disulfide Bonds
Catalog No.
- PF213-0.25-EX, PF213-2ML-EX, PF213-10ML-EX, PF213-50ML-EX
Supplements – Chaperones –
These are supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.
Supplements – Forming disulfide bonds –
This is supplement for synthesizing proteins containing disulfide bonds in an active form.