|PF004-0.5-EX||GroE Mix||500 µL for reaction||–||
GroE 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.
GroE Mix (#PF004-0.5-EX) is constituted of highly purified GroEL (known as Hsp60) and GroES (working in conjunction with GroEL) from E.coli with the optimized ratio.
GroE Mix works very well with PUREfrex®2.0 (#PF201-0.25-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 GroE Mix
After protein synthesis in 37 °C for 4 hrs, the synthesized protein was centrifuged and analyzed by SDS-PAGE. In the results of adding GroE Mix to PUREfrex®, insoluble proteins became to be detected in the soluble fractions.
|GroE Mix||12.5 μL||20µM GroEL, 14-mer form, and 40µM GroES, 7-mer form, 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 GroE Mix on ice.
- Mix Solution I, II, III and GroE Mix by vortex and centrifuge briefly to collect each solution at the bottom.
- Assemble the reaction mixture in a tube as follows.
Do not add GroE Mix at this step.
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 Template DNA X μL Total 19 μL
- Incubate the tube at 37 °C for 15 minutes.
（To prevent an inhibition of transcription, we recommend a pre-incubation before addition of GroE Mix.）
- Make a Two-fold dilution of GroE* Mix with Dilution buffer and add 1 µL to incubated tube in Step5.
- 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.
GroE Mix is developed for in vitro research only. GroE 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”