Q1: What do you recommend on the reaction condition for PUREfrex®?

Generally, we recommend reaction temperature of 37°C and a reaction time of 2 to 4 hours. However, the optimal conditions may differ depending on the proteins. For example, if synthesis at 37°C results in insolubilization or low activity, we recommend trying synthesis at other temperatures.

For more information, please see, "【Poster_MBSJ 2020】Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex®)"

Q2: Does reaction temperature affect?

YES.
Lowering the reaction temperature from 37℃ to 30°C or 25°C slows down the translation speed and generally reduces the amount of product. On the other hand, the folding is often slower than the translation, so slowing the translation may improve the solubility of the target protein.

Please see, "【Poster_MBSJ 2020】Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex®)"

Q3: Does pH affect?

YES.
The PUREfrex® reaction solution is buffered so that the pH is around 7.5. Therefore, when adding an acid solution or alkaline solution, adjust the pH of the reaction solution so that it is near neutral.

Q4: Does potassium ion affect?

NO.
If the final concentration of potassium ion in the PUREfrex® 1.0 reaction is in the range of 10-20 mM, there is no effect on protein synthesis.

Q5: Does magnesium ion and other divalent cations affect?

YES.
Regarding magnesium ion, if the final concentration in the PUREfrex® 1.0 reaction solution is only a few mM, there is little effect on protein synthesis. For other divalent cations, if the final concentration in the PUREfrex® 1.0 reaction solution is 10 mM or more, the amount of product is often reduced, so avoid using them as much as possible.

Q6: Does chelating agents such as EDTA affect?

YES.
The addition of a chelating agent to the PUREfrex® reaction solution will greatly affect the amount of product, so avoid using it as much as possible.

Q7: Does dimethyl sulfoxide (DMSO) affect?

NO.
If the final concentration of DMSO in the PUREfrex® 1.0 reaction is only a few percent, there is no effect on protein synthesis.

Q8: Does glycerol affect?

YES.
PUREfrex® basically contains a final concentration of 1.5% glycerol, under which conditions protein is synthesized. When glycerol is added, if the final concentration in the reaction solution is 5% or less, there is almost no effect on protein synthesis, but high concentrations of glycerol inhibit protein synthesis.

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