|PF003-0.5-EX||DnaK Mix||500 µL for reaction||–||
DnaK Mix is newly developed supplements for PUREfrex®2.0 to assist proper folding and improving solubility of the protein that needs molecular chaperones for proper protein folding, because PUREfrex®2.0 does 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®2.0 (#PF201-0.25-EX) and DS supplement (#PF005-0.5-EX) in a single 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.
Single tube reaction with PUREfrex® and DnaK Mix
After protein synthesis in 30 °C for 4 hrs, the activity of synthesized Luciferase was compared. As a result of adding DnaK Mix to PUREfrex, Luciferase productivity in active form was increased.
|DnaK Mix||25 μL||100µM DnaK, 20µM DnaJ and 20µM GrpE from E.coli in 30% glycerol buffer (No tags)||-80 °C*2|
|Dilution buffer||500 μL||30% glycerol buffer||-20 °C|
The amounts given are for a 20 µL reaction with PUREfrex®2.0. For scaling up the reaction, adjust the volume of reagents accordingly.
- Thaw Solution I by incubation at 30°C for 1 minute, and then cool on ice.
- Thaw Solution II, III and DnaK Mix on ice.
- Mix Solution I, II, III and DnaK Mix 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 6-X μL Solution I 10 μL Solution II 1 μL Solution III 2 μL DnaK Mix* 1 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.
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”