Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.2 Microplate Enzyme Activity Assays5.2.2 Microplate Glutathione S-Transferese AssayPage 1 of 25.2.2 Microplate Glutathione S-Transferase AssayWilliam G. BrogdonIntroductionElevated glutathione s-transferase (GST) activity has been associated with resistance to DDT. Usersshould be familiar with the contents of Microplate Enzyme Assays Introduction before proceeding.Materials• Acetone• Purified water• Reduced glutathione (e.g. Sigma G4251)• 0.25 M KPO 4 buffer (prepared as described in Microplate Enzyme Assays Introduction)• Brown glass storage bottlesReagent preparationGST solution1. Dissolve 61 mg reduced glutathione in 100 ml KPO4 buffer.2. Store at 4° C for up to 3-4 dayscDNB solution1. Dissolve 20 mg 1-chloro-2,4'-dinitrobenzene (cDNB) in 10 ml acetone.2. Add 90 ml 0.25 M KPO 4 buffer.Protocol1. To the plate on which you have previously added the 100 µl of mosquito homogenates (seeMicroplate Enzyme Assays), add 100 µl of 0.25 M KPO 4 buffer in three negative control wells on thelower right corner of the plate.2. To each well, add 100 µl reduced glutathione solution.3. To each well, add 100 µl cDNB solution.4. Read plate immediately (T 0 ) with microplate reader using 340 nm filter.5. Read plate at 5 minutes (T 5 ).6. Subtract the T 0 reading from the T 5 reading and use this for your statistical analysis.