Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance Monitoring5.2 Microplate Enzyme Activity Assays5.2.4 Microplate Oxidase AssayPage 2 of 22. Prepare two dilutions of the above for positive controls: 1:55 (i.e. 22 µl stock, 1.2 ml KPO 4 buffer) and1:110 (i.e. 11µl cytochrome-stock, 1.2 KPO 4 buffer).3. Use fresh.Protocol1. To the plate containing the mosquito homogenates (see Microplate Enzyme Assays Introduction),add 100 µl of KPO 4 to the negative and positive sample wells.2. Add 100 µl of the cytochrome-C positive control to three wells at the lower right side of the plate –undiluted and the two dilutions prepared above. 33. Add 200 µl TMBZ solution to each well.4. Add 1 drop (or 25 µl) 3% hydrogen peroxide (H 2 O 2 ) to each well.5. Incubate for 5 minutes.6. Read with microplate reader at 620 nm.3 Cytochrome-C is very photo labile. Make sure it is the last chemical you add to your oxidase platesbefore adding the TMBZ and H 2 O 2 .