I am looking for methods to make sure that all Non Structural Carbohydrates are being sampled in leaves following the NAD+/NADH technique. In order to do so, what is the best way to "dissolve" leaves powder in order to extract most of the NSC from cells and their surroundings.
I use perchloric acid to extract soluble sugars, as it immediately inactivates all enzymes which may metabolize the sugars you are interested in. The advantage compared to Ethanol is that fewer pigments get extracted together with the sugars, leading to a lower background.
1. Freeze your plant sample in liquid nitrogen and grind it using a ball mill, add 1 ml of 0.7M Perchloric acid per 100 mg of fresh weight to the powder, and mix again by using the ball mill.
Alternatively, you can add the plant and 1 ml 0.7 Perchloric acid per 100 mg Fresh weight to a glass homogeniser and extract the soluble compounds this way.
2. Spin down at 3000g for 5 min at 4°C
3. Transfer supernatant to a new tube and neutralise the perchloric acid by adding neutralisation buffer (2M KOH 0.4M MES). The volume of neutralisation buffer will be approximately one third of the supernatant, depending on the plant sample. I usually check the pH of the first samples with a pH stick to determine the exact ratio. The pH should be between 6 and 7.
4. Remove the precipitated potassium perchlorate by centrifugation (10 min, 10000g, 4°C).
5. Transfer supernatant to an new tube and store at -20°C.
You can then use an aliquot for the determination of the soluble sugars.
The extraction method was first published by Fulton et al 2008: http://www.plantcell.org/content/20/4/1040.short . Although there the authors used HPLC for quantification of the sugars I also use the NAD+/NADH method if I don't need to quantify maltose.
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