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Assay Workflow

Odyssey CLx with a plate
  1. Remove cell culture medium.
  2. 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.

  3. 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.

  4. Shake for 5 min at room temperature.
  5. 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).
  6. Optional: Add 25 μL stop buffer to each well (final concentration 0.33 M).
  7. Should you add stop buffer? Things to consider:

    Odyssey Sa
    • 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.
  8. Image plate with an Odyssey® Infrared Imaging System.

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