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CONTENTS
Overview
DnaK Mix is newly developed supplements for PUREfrex® series (Cell-free protein Synthesis Kits) to assist proper folding and improving solubility of the protein that needs molecular chaperones for proper protein folding, since PUREfrex® series (Cell-free protein Synthesis Kits) do not contain molecular chaperones.
DnaK Mix is consisted of highly purified DnaK, DnaJ and GrpE from E.coli with optimized ratio. DnaK known as Hsp70 has ATPase activity and is stimulated by co-chaperones, DnaJ and GrpE. DnaJ facilitates the ATPase activity of DnaK and could bind to a hydrophobic region of synthesized protein. GrpE stimulates ADP/ATP exchange rate of DnaK.
DnaK Mix works very well with PUREfrex® series (Cell-free protein Synthesis Kits) and DsbC Set (Supplement for forming disulfide bonds) in a same tube for protein synthesis reaction, which could lead to the preparation of your protein in proper folding with good solubility according to the character of your protein.
Figure. Effect of DnaK Mix on the activity of synthesized luciferase.
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 DnaK Mix as supplement to PUREfrex® reaction mixture if the solubility or activity of the synthesized protein is low.
Protocols
The amounts given are for 20 µL reaction with PUREfrex®2.0. 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, III and DnaK Mix on ice.
3. Mix Solution I, II, III and DnaK Mix 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 | 6-X μL |
| Solution I | 10 μL |
| Solution II | 1 μL |
| Solution III | 2 μL |
| DnaK Mix *1 | 1 μL |
| Template DNA |
X μL |
| Total | 20 μL |
|---|
5. Incubate the tube at 37 °C for 2 - 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.
- The optimum concentration of DnaK Mix depends on a protein. Please use "Dilution buffer" (in the kit) to dilute the DnaK mix.
Specifications
Kit components <DnaK Mix>
| Reagent | Quantity for 500 μL rxn |
Quantity
for 2 mL x 5 rxn |
Description | Storage *1 |
|---|---|---|---|---|
| DnaK Mix |
25 µL | 100 μL x 5 | 100µM DnaK, 20µM DnaJ and 20µM GrpE from E.coli in 30% glycerol buffer (No tags) |
-80 °C *2 |
| Dilution buffer |
500 µL | 500 μL x 1 | 30% glycerol buffer |
-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.
Datasheet
PF003-0.5-EX
DownloadsPF003-10-EX
DownloadsSDS
US
DownloadsEU
DownloadsNote
Dnak Mix is developed for in vitro research only. Dnak Mix 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® series.
Cell-free Protein Synthesis Kits
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.