#PF003-10ML-EX
For 2 mL x 5 Reaction
PUREfrex® is NOT included.

in vitro research use only
Store at -80ºC before opening
GeneFrontier Corporation
www.genefrontier.com

May 2023

1. Overview

2. About PUREfrex®

DnaK Mix is a newly developed supplement ofPUREfrex® to assist proper folding and solubility of yourprotein.
PUREfrex® is a cell-free protein synthesis reagent but has
no molecular chaperones (See next page).
When your protein of interest needs molecularchaperones for proper protein folding, DnaK Mix could bea solution for that.
DnaK Mix is constituted of highly purified DnaK, DnaJand GrpE from E.coli with the optimized ratio.
DnaK known as Hsp70 has ATPase activity and isstimulated by co-chaperones, DnaJ and GrpE.
DnaJ facilitates the ATPase activity of DnaK and could
bind to a hydrophobic region of protein.
GrpE stimulates ADP/ATP exchange rate of DnaK.
DnaK Mix works very well with PUREfrex®
(#PF001-0.25-EX or #PF201-0.25-EX) (and with DsbC Set(#PF005-0.5-EX)) in a single tube for protein synthesis
reaction, which could lead to the preparation of yourprotein in proper folding with good solubility.

PUREfrex® is a reconstituted cell-free protein synthesis
kit which GeneFrontier has developed based on the PURE
system technology. The PURE system is a cell-free protein
synthesis system, which has originally developed byProfessor Takuya Ueda at the University of Tokyo, and is
c o n s i s t e d o f o n l y p u r i fi e d f a c t o r s n e c e s s a r y f o rtranscription, translation and energy regeneration (Ref. 1).
The target protein is synthesized by adding the templateDNA (or mRNA) to the reaction mixture. PUREfrex® isconsisted of only purified factors, therefore it enables toadjust the composition of the reaction mixture.
PUREfrex® is raised in the purity by improving thepreparation methods of ribosomes, tRNAs and all proteinsin the reaction mixture compared with the original PURE
system (Ref. 2). As the result, the contaminating
lipopolysaccharide from E. coli is reduced below 0.1 EU per
1 µL of reaction and other contaminants, such as RNase
and β-galactosidase, are also reduced.
In the PUREfrex®, all proteins have no tags forpurification or detection, therefore the target protein
would be synthesized and purified by any tag.

SHARP Kashiwa Building, 4F
273-1 Kashiwa Kashiwa-shi,Chiba 277-0005 JapanReferences) 1. Shimizu et al. (2001) Nat. Biotecnol., vol. 19, p. 751
2. Shimizu et al. (2005) Methods, vol. 36, p. 299

100 µL x 5

100µM DnaK, 20µM DnaJ and 20µM GrpE in 30% glycerol
bufferStore at -80ºC *1

Dilution Buffer (Clear)

DnaK Mix is worked with PUREfrex® (#PF001-0.25-EX,#PF201-0.25-EX) or DsbC Set (#PF005-0.5-EX) in one tube.
For example, 20 µL of reaction is assembled as below.

500 µL x 1

1. Thaw Solution I by incubation at 30ºC for 1 minute,and then cool on ice.

Store at -20ºC

2. Thaw Solution II, III and DnaK Mix on ice.

30% glycerol buffer

5. Incubate the tube at 37ºC for 2-4 hours.
6. Analyze the synthesized product.

3. Mix Solution I, II, III and DnaK Mix by vortex andcentrifuge briefly to collect each solution at the bottom.
4. Assemble the reaction mixture in a tube as follows.
(Add the template DNA to 0.5-3 ng/µL per 1 kbp)

*1)
For storage at -80ºC, the rest of solution should be frozen rapidly inliquid nitrogen or dry ice/ethanol. Divide into aliquots, if necessary,and avoid refreeze and thaw as much as possible.

or administration to animals including human and should
not be used as food or cosmetics etc.
To avoid the contamination of nuclease, nuclease-freetreated water, reagents and materials should be used. We
also recommend wearing gloves and mask.
For information concerning commercial use of DnaK
Mix, please contact GeneFrontier.
e-mail : purefrex@genefrontier.com【Outside Japan】【JAPAN】

Carlsbad, CA 92010, USA

KOTO-KU. TOKYO 135-0016, JAPAN2792 Loker Ave West, Suite 101
email: info@cosmobiousa.com

Phone/FAX: (+1) 760-431-4600
URL: www.cosmobiousa.com

Store at -80ºC before opening

DnaK Mix (Orange)

DnaK Mix is developed for in vitro research use only.
DnaK Mix should not be used for the therapy, diagnostic

Water
Solution I
Solution II
Solution III
DnaK Mix *2
Template DNATotal#PF001
7-X µL
10 µL
1 µL
1 µL
1 µL
X µL
20 µL

#PF201
6-X µL
10 µL
1 µL
2 µL
1 µL
X µL
20 µL*2)
The optimum concentration of DnaK Mix depends on protein ofinterest. Please use Dilution buffer for dilution of DnaK Mix.

TOYO EKIMAE BLDG. 2-20, TOYO 2-CHOME,
Phone: +81-3-5632-9610
FAX: +81-3-5632-9619

URL: https://www.cosmobio.co.jp/