|Product||Catalog No.||Quantity||SDS*||Price (JPY)||Instruction|
|PUREfrex®2.0||PF201-0.25-EX||250 µL for reaction||–||
|PUREfrex®2.0||PF201-0.25-5-EX||1250 µL for reaction||–||
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We modified the preparation methods of all components that were purified from E.coli and optimized the factors’ composition. As a result of this modification, PUREfrex®2.0 achieved 2-10 times higher protein productivity than PUREfrex®1.0.
By improving the purification process of components of PUREfrex®, contamination of RNase and β-galactosidase are greatly reduced, in addition to that, lipopolysaccharide (LPS) is also reduced to around 0.1 EU per 1 µL of reaction mixture. All proteinous components of PUREfrex®2.0 have no tags for purification and detection. It allows to fuse your protein with any tag to purify the product.
- 30 µL of reaction was incubated at 37 °C for 2-4 hrs.
- 270 µL of Binding buffer (50mM Tris-Cl pH8, 500mM NaCl, 20mM MgOAc*) was added to the above reaction.
* To prevent the dissociation of ribosomes
- 30 µL of 5% Nickel Magnetic Beads (PureProteome™ ;Millipore) suspension in Binding buffer was added to the above.
- Incubated for 1-2 hrs at room temperature.
- Washed the Nickel Magnetic Beads with Binding buffer.
- Eluted His-tagged protein by Elution buffer (50mM Tris-Cl pH8, 500mM NaCl, 100mM imidazole).
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.
Supplements – Chaperones –
This is 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.
|Solution I||125 μL||Amino acids, NTPs, tRNAs and substrates for enzymes etc.||-20 °C|
|Solution II||12.5 μL||Proteins in 30 % glycerol buffer||-20 °C or -80 °C *2|
|Solution III||25 μL||Ribosome (20 μM)||-80 °C *2|
|10 μL||PCR product containing a gene encoding dihydrofolate reductase (DHFR) from E.coli as a positive control. (20ng/μL)||-20 °C|
The amounts given are for 20 µL reaction. For scaling up the reaction, adjust the volume of reagents accordingly.
- Thaw Solution I by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
- Thaw Solution II and III on ice.
- Mix Solution I, II and III respectively by vortex and centrifuge briefly to collect each solution at the bottom.
- Assemble the reaction mixture in a tube as follows. Add the template DNA to 1 – 3 ng/µL per 1 kb.
Water 7-X μL Solution I 10 μL Solution II 1 μL Solution III 2 μL Template DNA X μL Total 20 μL
- Incubate the tube at 37 °C for 2 – 4 hours on a heat block or a water bath.
- Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.
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”