|Catalog No.||Product||Quantity||SDS||Price (JPY)||Instruction|
|PF006-0.5-EX||PDI Set||500 µL for reaction||US
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“PDI Set” is a supplement by adding to PUREfrex® for synthesis proteins with correct disulfide bonds 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.
PDI Set includes oxidized glutathione (GSSG), human PDI (protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).
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.
DsbC Set (#PF005-0.5-EX)
This is supplement for PUREfrex® which includes disulfide isomerase from E.coli for synthesizing proteins containing a disulfide bonds in active form.
|GSSG*2||25 μL||Oxidized glutathione (60 mM)||-20 °C|
|PDI*3||25 μL||Human protein disulfide isomerase with no tags (200 µM)||-80 °C*4|
|Ero1α*3||25 μL||Human ER oxidoreductin-1 to reoxidize PDI with no tags (5 µM)||-80 °C*4|
|Dilution buffer||500 μL||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) Standard final concentration of GSSG is 1 – 3 mM with 4 mM GSH (reduced glutathione) in PUREfrex® 2.1 (#PF213). We recommend to check the optimal concentration of GSSG because it depends on the target protein and the kind and concentration of reducing agent.
*3) Standard final concentration of PDI/Ero1α is 1/0.025 – 10/0.25 µM. We recommend to check the optimal concentration of PDI and Ero1α because it depends on the target protein. Please use attached Dilution Buffer for diluting PDI and Ero1α.
*4) For storage at -80°C, the rest of solution should be frozen rapidly in liquid nitrogen or dry ice/ethanol. Please divide into aliquots, if necessary, and avoid refreeze and thaw as much as possible.
This is a standard protocol for synthesizing proteins containing disulfide bonds using PDI Set and PUREfrex® 2.1 (#PF213). For example, please assemble 20 µL of reaction mixture as below, in which the final concentration of each reagent is 0.5 mM Cysteine, 4 mM GSH, 1 mM GSSG and 10 µM PDI.
- Thaw Solution I, Cysteine, GSH and GSSG by incubation at room temperature or 37 °C for 1 minute for completely dissolving, and then cool on ice.
- Thaw Solution II, III and PDI on ice.
- Mix each solution by vortex and centrifuge briefly to collect each solution at the bottom.
- Dilute GSSG 3-fold with water.
- 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 20 mM GSSG *2 1 μL Solution II 1 μL Solution III 2 μL PDI 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.
PDI Set is developed for in vitro research only. PDI 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”