Analytical Techniques

This chapter presents analytical techniques for protein analysis and measurement of starch and sucrose in leaves. When measuring any metabolite, it is important to stop leaf metabolism immediately and to prevent subsequent action of endogenous enzymes on metabolites. The best method of stopping metabolism is to freeze the leaf in liquid N2, preferably by freeze-clamping. The plant material is rapidly clamped between the two discs, which causes both rapid cooling and effective disruption of the tissue. Other freezing mixtures may be employed along with boiling alcohol, although the latter has the disadvantage that samples must be dried down. Measurement of any metabolite or compound in plants should be accompanied by a careful check that it has not been degraded or absorbed into the material collected in the pellet during the killing and extraction procedure. Sucrose provides an example of such liability. The best check is to prepare and extract duplicate samples of tissue, adding a measured amount of the metabolite to a pestle and mortar. The amount of the compound added should be comparable to that present in the tissue.