|Product||Catarog No.||Quantity||SDS*||Price (JPY)||Instruction|
former DS supplement
|PF005-0.5-EX||500 µL for reaction||–||
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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.
PUREfrex® 2.1 (#PF213-0.25-EX)
This is cell-free protein synthesis kit which can change a type and amount of reducing agent freely.
DnaK Mix (#PF003-0.5-EX)
This is supplement for PUREfrex® which is molecular chaperone to be aggregate-prone protein in a soluble form.
PDI Set (#PF006-0.5-EX)
This is supplement for PUREfrex® which includes disulfide isomerase from human for synthesizing proteins containing a disulfide bonds in active form.
|GSSG||25 μL||Oxidized glutathione (60 mM)||-20 °C|
|DsbC||25 μL||E.coli DsbC (320 µM*2) (No tags)||-80 °C *3|
|Dilution buffer||500 μL||30% glycerol buffer||-20 °C|
This is a standard protocol for synthesizing proteins containing disulfide bonds.
Each solution of DsbC Set (#PF005) and PUREfrex® 2.1 (#PF213) are mixed together in a same tube.
- Thaw Solution I, Cysteine, GSH and GSSG by incubation at room temperature or 37 °C for 1 minute completely, and then cool on ice.
- Thaw Solution II, III and DsbC on ice.
- Mix each solution 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 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
- 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.
- Analyze the synthesized products. Please add the same amount of H2O to the reaction for the sample of SDS-PAGE.
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”