Protocol for the detection of proteolytic activity in female Aedes aegypti mosquito midgut extracts using fluorogenic AMC protease substrates

Female Ae. aegypti mosquitoes utilize midgut proteolytic enzymes to digest blood meal proteins, but activity detection has largely depended on the use of chromogenic substrates. Here, we present a protocol for the detection of proteolytic activity in female mosquito midgut extracts using fluorescent 7-amino-4-methylcoumarin (AMC) protease substrates. We describe steps for midgut tissue and substrate preparation, as well as proper dilutions for easy fluorescent detectable readouts in 30 μL reactions. This approach saves time and minimizes sample usage. For complete details on the use and execution of this protocol, please refer to Ramirez et al.¹.