Mag-Indo-1 tetrapotassium salt

Mag-Indo-1 tetrapotassium salt can be used to determine the free Mg²⁺ concentration in the cytoplasmic solutions^[1].
Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)^[1].
1. Calibrating solutions containing various concentrations of Mg²⁺ are prepared by mixing different ratios of the two stock solutions: one contains (mM) 150 KCl, 0.1 EGTA and 5 Hepes (pH 7.2 with KOH), and the other contains 100 MgCl₂, 0.1 EGTA and 5 Hepes (pH 7.2 with KOH).
2. The calibration curve is constructed using calibrating solutions containing 1 μM Mag-Indo-1 tetrapotassium salt.
3. The 'Mg²⁺' calibrating solution contained (mM): 130 KCl, 4 EDTA and 5 Hepes (pH 7.2 with KOH).
4. The relationship between the background-corrected value of the fluorescence ratio (R) and the Mg²⁺ concentration is fitted with the following theoretical equation.
[Mg]=K (R-R_min)/(R_max-R), where [Mg] is the concentration of free Mg²⁺ ion, R_min is the R value at 0 [Mg²⁺], and R_max is the R value at saturating Mg²⁺. The curve fitting gave R_min= 0.053, R_max= 1.57, and K= 5.1 mM.
Note: the specific curve can refer to the reference.