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^®)”