Contact: Dr. Christos Chinopoulos MD, PhD chinopoulos.christos@eok.sote.hu
Limitations of the method


As with any other method, the present one has its own limitations. These are:

Limitation #1: Estimation of ADP-ATP exchange rate using MgG fluorescence cannot be used in the presence of a high concentration of  Ca2+ in the medium; MgG is sensitive to Ca2+, see 'Why Magnesium Green?'.

Limitation #2: Estimation of ADP-ATP exchange rate using MgG fluorescence cannot be used simultaneously with other fluorophores with spectral overlap to that of MgG.

Limitation #3: Estimation of ADP-ATP exchange rate using MgG fluorescence cannot be used if your mitochondria exhibit a relatively high Mg2+ transport activity (rare property of Arabidopsis and yeast mitochondria). Of course all types of mitochondria exhibit Mg2+ transport mechanisms, but unlike those reported in Arabidopsis and some yeasts, Mg2+ flux is negligible). 

Limitation #4: Nor FCCP neither CCCP can be used while estimating ADP-ATP exchange rate using MgG fluorescence; these compounds quench MgG fluorescence (or any other fluorophore based on the fluorescein structure, such as Calcium Green). you can use SF6847, also known as Tyrphostin 9 or Malonaben (see: The interaction of highly active uncouplers with mitochondria by Terada H, 1981, Biochim. Biophys. Acta. 639:225-242). SF6847 can be purchased from here:
http://www.enzolifesciences.com/BML-EI215/tyrphostin-9/
50 mg costs 62 EUR, 177.0538  milliliters for 1 mM stock, enough for 354,106 experiments (0.5
mM or less is needed for complete collapse of mitochondrial membrane potential).