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easily be used for measurements of free [Ca 2 þ ] in experimental solutions or
biological fluids. Indeed, for those studying Ca 2 þ -dependent processes, there is
no practical or economical reason why Ca 2 þ electrodes should not be used as
routinely as pH electrodes.
III. Methods
A. Preparation of Minielectrodes
Ca 2 þ -selective minielectrodes can be prepared by dipping polyethylene (PE)
tubes (typically
5 cm) in a membrane solution (see composition below). We
have tried other types of tubing but polyvinyl chloride (PVC) tubing appears to
absorb ETH 129 from the Ca 2 þ -selective membrane, resulting in a faster loss of
sensitivity as compared to the PE tubing. On the other hand, materials such as
Teflon tubing absorb little ETH 129 but the PVC membrane does not adhere well
to the tubing. As a result, the electrodes are more easily damaged, although they
may have a longer lifetime if handled with care.
The dimensions of the tubing vary from a diameter less than 1 to
3 mm.
Electrodes prepared with the membrane solution described below result in Ca 2 þ -
selective membranes that are a few hundred micrometers thick. With diameters
larger than 5 mm, the Ca 2 þ -selective membrane bursts more easily during
handling, but this problem may be overcome by inserting a ceramic plug into the
tubing, before dipping it in the membrane solution as described by Orchard et al.
(1991) . For general purposes we have used inner electrode diameters of 1.67 mm
(PE 240, Clay Adams).
After dipping the PE tubing in the membrane solution, the Ca 2 þ -selective
membrane is allowed to dry overnight. Then the electrode is filled with an appro-
priate filling solution, which should correspond to experimental conditions (see
below). After filling the electrode, it is allowed to equilibrate for at least 3 days in a
glass vial containing the filling solution (but see below).
1. Preparation and Use of the Ca 2 þ -Selective Ligand
The Ca 2 þ -selective membrane can be prepared as described by Schefer et al.
(1986) (all from Fluka/Sigma-Aldrich; Table I ).
ETH 129 is dissolved in N-phenyl-octyl-ether (NPOE) under vigorous stirring in
a small glass vial (Solution 1). At the same time, PVC is dissolved in the THF;
when completely dissolved, potassium TCPB is added (Solution 2). When the
components of Solutions 1 and 2 are completely dissolved, the two solutions are
mixed, and the membrane solution is ready to use, or can be stored in a glass vial,
closed with a Teflon screwcap and protected from light. If THF evaporates from
the membrane solution during storage, a small amount of THF can be added to
achieve the desired viscosity of the membrane solution.
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