- Remove cell culture medium.
- Optional: wash cells once with PBS.
Note: If the cells are attached loosely to the plate, this wash step may cause cell loss and should be omitted.
- Add 50 μL DDAOG Reaction Solution to each well of the 96-well plate.
Note: if using a different size of well or reaction vessel, you may need to adjust the reaction solution volume.
- Shake for 5 min at room temperature.
- Incubate for 20-60 min at 37 °C (the optimal incubation time for your experiment can be determined by imaging the plate every 15 min).
- Optional: Add 25 μL stop buffer to each well (final concentration 0.33 M).
Should you add stop buffer? Things to consider:
Image plate with an Odyssey® Infrared Imaging System.
- While you are scanning one region of the plate, the reaction will continue to progress in other wells. To most accurately measure the enzymatic activity in ALL wells at a given time point, you must add stop buffer.
- If necessary, you can stop the reaction with stop buffer and save the plate at room temperature to be imaged later. Protect from light during storage.
- When determining the optimal incubation time for your reactions, you must let the reaction proceed and cannot use stop buffer.
- Addition of stop buffer may decrease signal intensity, because it dilutes the reaction solution in the well.