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Quantifying calcium - Calibration & Tips

Experimental Tips for Calibration:

EGTA or other chelators are added to the extracellular medium during the measurement of Fmin, though using an intracellular chelator as BAPTA is highly recommended to ensure zero free calcium intracellular concentration.

Fmax can be obtained using 1 mM extracellular concentration or with a [Ca2+] bigger than 10·Kd.

F has to be measured for a range of calcium solutions of known concentration. This solutions are usually prepared using a mixture of CaEGTA/EGTA at suitable rates, but special care must be taken to avoid artefacts: free calcium concentration in CaEGTA/EGTA mixtures is function of the same factors that affect other calcium chelators, i.e. temperature, ionic strength, pH, etc.

Equation 1 can be used to obtain [Ca2+]free for a known Kd value of CaEGTA.


[Ca2+]free = Kd [CaEGTA] / [K2EGTA]

Equation 1.

A typical Kd value for EGTA at 37ºC, pH 7,2 in 0,1 M KCl is 108 nM (a range of Kd values at different pH and temperature is described at Methods Enzymol. 172, 230 (1989)). A small variation in temperature or pH might change Kd value quite a lot (at 20ºC, Kd is 150 nM, while at pH 7,30 and 37ºC Kd is 69 nM), so corrections have to be made if our experimental conditions are different from those tabulated (Methods Cell Biol. 40, 3 (1994)).

See an example of a calibration experiment.



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