Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

6 Yazawa
Fig. 2. Shop drawing for the Reichard-type flow-dialysis cell. Left, the upper chamber;
right, the lower chamber. Dimensions are shown in millimeters. The flow-dialysis
cell is made of Teflon.
The membranes are heated to approx 80°C on the boiling water bath for approx
30 min once in 1 mM ethylenediaminetetracetic acid (EDTA), twice in deionized
water, then in 1% acetic acid, and twice in deionized water. The washed membranes
in deionized water are stored in the refrigerator.
9. Buffer solution: A suitable buffer solution (approx 500 mL) is prepared using
deionized distilled water or a Milli-Q water. The buffer solution is preferable to
contain 50 mM or higher concentration of NaCl or KCl. It should be degassed
sufficiently with stirring under reduced pressure using an aspirator immediately
before the measurement, which is essential for preventing formation of bubbles
in the buffer chamber.
10. Stock solutions of Ca 2+ : The stock solutions of Ca 2+ used in the Ca 2+ titration are
prepared from CaCl 2·2H 2O by weight and stored in plastic bottles (see Note 5).
The Ca 2+ concentration can be determined by atomic absorption spectrometry.
11. Radioactive Ca 2+ ( 45 Ca 2+ ): The radioactive isotope available for Ca, 45 Ca, is a
β-emitting nuclide with a half-life of 164 d and a maximum energy of 0.254 MeV.
We purchase 45 CaCl 2 solution with sufficiently high specific radioactivity from