Calcium-Chelator Solution for Metal–Chelate Buffer System

• This recipe is for a 1 m Ca²⁺, 1 m EGTA solution. Use the same procedure to make stock solutions of 1 m Ca²⁺ with 1 m EDTA, HEDTA, or NTA.

Use the pH titration method (Tsien and Pozzan 1989) to make 10 mL of a 1 m Ca²⁺, 1 m EGTA solution. Weigh out 3.84 g EGTA free acid. Add 1.2 g KOH pellets, 0.95 g CaCO₃ (9.5 mmol), and 6 mL Chelex-treated ultrapure H₂O, stirring vigorously and heating slightly to get EGTA into solution. Add 10 n KOH, 10 µl at a time, until the pH is between 7 and 8. Add 20 µL 1 m CaCl₂ (0.2 mmol) and record the decrease in pH. Add ∼30 µL 10 n KOH to re-adjust the pH so that it stays between 6.5 and 8. Repeat the addition of CaCl₂ and KOH until the magnitude of the pH change after each addition of CaCl₂ decreases by 50%. Add 10 n KOH to adjust pH to ∼7.4. Use a volumetric flask to bring the total volume up to 10 mL with Chelex-treated ultrapure H₂O. Using this method, the Ca²⁺ and EGTA concentrations should be within 0.5% of each other.

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