2.4. Trehalose assay

1. After taking the glucose readings, pipette 0.5 µl of the trehalase enzyme into each cuvette or well of the microplate including the wells containing the glucose standards (Flatt et al., 2008; Ishikawa et al., 2009).

2. Slowly shake the plate on a rocking platform for 1 min.

3. Centrifuge for 2 min.

4. After centrifugation, use a piece of Parafilm to seal the cuvettes or microwell plate.

5. Incubate overnight at 37ºC overnight.

6. Centrifuge again.

7. Read absorbance as described above for glucose (step 11 in 2.3)

8. Calculate the trehalose concentration as:

Trehalose concentration

 

The trehalose concentration is calculated as the reading of the proper reaction minus the prior determined glucose concentration. The term is then multiplied by the molecular weight of trehalose (342.3) and, as trehalose is split by trehalase into two glucose molecules, the entire term is finally divided by 2 x the molecular weight of glucose (180.2).