It is very critical. Waiting too long for analysis post transfection can lead to loss of peak expression. The optimal time for analysis is related to two factors: stability of protein being expressed and transfection method. For a short-lived protein (like luciferase) analysis should be done at 6-18 hours after Nucleofection™. For a more stable protein, such as GFP, one can wait to start analysis for up to 24 hours or longer post Nucleofection. As Nucleofection™ delivers DNA directly to nucleus at the time the pulse is delivered, expression will start much earlier than with other transfection methods. The best practice is to always perform a time point analysis when expressing a new protein. Further information on this topic you can find in the attached technical reference guide