The recommended incubation temperate range is 20–40˚C. For incubations longer than 1 hour, we recommend 25˚C. The incubation time is substrate dependent and varies based on the reason for proteolysis. Typically, to eliminate enzyme activity, a 5–10 minute incubation is adequate. However, it may take longer if cells need to be lysed, nucleosomes need to be degraded, histones need to be cleaved, or if other proteins are present.