DsbC Set

Supplements – Forming disulfide bonds –

DsbC Set


Size Price (USD)
500 µL reaction $88.00


Size Price (USD)
2 mL x 5 reaction $1400.00

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"DsbC Set" is the supplement by adding to PUREfrex® for synthesizing proteins containing a disulfide bond in an active form.

Formation of a disulfide bond is an important process for folding and stability of secretory proteins such as enzymes or antibodies. A disulfide bond is usually formed by the oxidation of sulfhydryl group (SH-) of adjacent cysteine residues. Therefore, the formation efficiency of a disulfide bond depends on redox state. Additionally, disulfide bond isomerase which can catalyze the exchange of disulfide bridges may be also required for a correct pairing of cysteines.

DsbC Set is constituted of highly purified DsbC from E.coli known as disulfide bond isomerase, which can catalyze the disulfide bridge exchange, and GSSG (oxidized Glutathione) to make an oxidized environment.

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 DsbC Set as supplement to PUREfrex® reaction mixture if the solubility or activity of the synthesized protein is low.


This is a standard protocol for synthesizing proteins containing disulfide bonds.
Each solution of DsbC Set and PUREfrex®2.1 are mixed together in a same tube.

1. Thaw Solution I, Cysteine, GSH and GSSG by incubation at room temperature or 37 °C for 1 minute completely, and then cool on ice.

2. Thaw Solution II, III and DsbC on ice.

3. Mix each solution 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 5-X μL
Solution I *1 8 μL
10 mM Cysteine
1 μL
80 mM GSH 1 μL
60 mM GSSG 1 μL
Solution II 1 μL
Solution III 2 μL
DsbC (80 μM) *2 1 μL
Template DNA *3 X μL
Total 20 μL

5. Incubate the tube at 37 °C for 2 - 6 hours with heat block or water bath.
Protein synthesis reaction is almost done until 6 hours, but some proteins require longer incubation (e.g. 24 hours) to form disulfide bonds between the correct pair of cysteine residues.

6. Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.

  1. This volume is different from Solution I of PUREfrex®2.0.
  2. DsbC is diluted with Dilution buffer included in DsbC Set.
  3. Please visit the template DNA preparation site.


Kit components   <DsbC Set>

Reagent Quantity
for 500 μL rxn
for 2 mL x 5 rxn
Description Storage *1
GSSG 25 µL 100 µL x 5 Oxidized glutathione (60 mM) -20 °C
DsbC 25 µL 100 µL x 5 E.coli DsbC (320 µM *2) (No tags) -80 °C *3
Dilution buffer
500 µL 500 µL x 1 30% glycerol buffer
-20 °C
  1. Indicated temperature shows storage temperature after opening the kit. Please store the kit at -80 °C before opening.
  2. It's same concentration as 7.5 mg/mL described in former instruction.
  3. 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.








DsbC Set is developed for in vitro research only. DsbC Set 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"


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


for Basic Research and Customized kit

Catalog No.

  • PF001-0.25-EX, PF001-0.25-5-EX


for Syntesis Proteins

Catalog No.

  • PF201-0.25-EX, PF201-0.25-5-EX


for Forming Disulfide Bonds

Catalog No.

  • PF213-0.25-EX, PF213-0.25-5-EX

Supplements – Chaperones –

These are supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.

DnaK Mix


Catalog No.

  • PF003-0.5-EX

GroE Mix


Catalog No.

  • PF004-0.5-EX

Supplements – Forming disulfide bonds –

This is supplement for synthesizing proteins containing disulfide bonds in an active form.


Human isomerase, Ero1, GSSG

Catalog No.

  • PF006-0.5-EX


Can I synthesize eukaryotic proteins using the PUREfrex® kit?
The PUREfrex® is a reconstituted cell-free protein synthesis kit composed of E. coli ribosomes and translation factors, but eukaryotic proteins from such as mammalian and plant can also be synthesized. The productivity of the target protein may depend on the property of nucleotide sequence encoding the target protein, such as GC contents or frequency of rare codon.
How much can I synthesize the target protein using the PUREfrex® kit?
It depends on the target protein.
Dihydrofolate reductase from E.coli can be synthesized with about 600 μg per mL of the reaction.
Can I synthesize proteins of more than 100 kDa?
We have synthesized the protein of about 120 kDa using PUREfrex®.
What do you recommend the reaction condition for PUREfrex®?
We recommend that the protein synthesis using PUREfrex® is carried out at 37 °C for 2-6 hours.
Can I synthesize and purify a tagged protein?
You can use any tag, because all of protein components of PUREfrex® have no tag for purification or detection. For example, the target protein with histidine tag can be purified with metal-chelating resin by applying reaction mixture directly, after synthesizing.
Is synthesized protein modified by glycosylation or phosphorylation?
There is no post-translational modification, because PUREfrex® is constituted by translation factors only.
Does PUREfrex® contain any molecular chaperones?
PUREfrex® does not contain any chaperones, but you can add chaperones, such as Hsp70.
GeneFrontier has been developing molecular chaperones suitable for PUREfrex® kit, DnaK Mix (#PF003-0.5-EX) and GroE Mix (#PF004-0.5-Ex).
Can I synthesize a protein containing disulfide bonds with PUREfrex®?
DsbC Set (#PF005-0.5-EX) and PDI Set (#PF006-05-EX) are supplements by adding to PUREfrex® for synthesizing proteins containing a disulfide bond in an active form. PUREfrex® 2.1 (#PF213-0.25-EX) is recommended to optimize the reaction conditions.
Can I synthesize a membrane protein with PUREfrex®?
In the most of the cases, a synthesized membrane protein may form aggregates. To obtain a membrane protein inserted into a lipid bilayer, you may add a lipid bilayer such as liposome to PUREfrex® when you synthesize the membrane protein.
Can I synthesize a radiolabeled protein with [35S] methionine or [3H] leucine?
A radiolabeled protein can be synthesized by adding radioisotope-labeled amino acids, such as [35S] methionine or [3H] leucine, to PUREfrex®. The amino acid content is 0.5 mM for PUREfrex®1.0 and 2 mM PUREfrex®2.0. Please consider that point and optimize conditions for your labeling.
Can I use any promoter besides T7 promoter?
We recommend the template DNA with T7 promoter, because PUREfrex® contains T7 RNA polymerase for transcription. When you use other polymerases, please generate template DNA with the suitable promoter for the selected polymerase.
I can’t get DHFR using DHFR DNA in the kit as positive control.
1. The kit may be inactivated for some reasons. To avoid the inactivation, please store the kit at suitable temperature and divide into aliquots to reduce freeze-thaw cycles as much as possible.

2. The kit may be contaminated by nuclease. To avoid a nuclease contamination, please use nuclease-free water, reagents and materials.
I can get DHFR using DHFR DNA in the kit. But I can’t get my protein of interest or can get it with very low amount.
1. The kit may be inactivated for some reasons. To avoid the inactivation, please store the kit at suitable temperature and divide into aliquots to reduce freeze-thaw cycles as much as possible.

2. The kit may be contaminated by nuclease. To avoid a nuclease contamination, please use nuclease-free water, reagents and materials.
Sometimes the plasmid is contaminated with RNase through a plasmid preparation process. So, please confirm that RNase is removed from the plasmid.

3. The construct of template DNA may be incorrect. It is necessary to contain the sequence of T7 promoter, ribosome binding site, initiation codon, and stop codon for the template DNA for PUREfrex®.

4. Secondary structure of transcripts may prevent the translation reaction. In this case, please optimize the template sequence to solve the problem of secondary structure.