Biology Reference
In-Depth Information
Table I
Preparation
PVC-based Ca
2þ
-selective
of
the
ligand
minielectrodes
Component
*
Amount
Solution 1
ETH 129
25 mg
N-phenyl-octyl-ether (NPOE)
451.5
m
l
Solution 2
Polyvinyl chloride (PVC)
250 mg
K
þ
-tetrakis chlorophenyl borate (TCPB)
12.9 mg
Tetrahydrofuran (THF)
5ml
*
Components can be obtained from Sigma-Aldrich (St. Louis, MO)
The filling solution used for the minielectrode depends on the experimental
solutions. Generally, the ionic composition should mimic the environment in
which measurements of Ca
2
þ
are planned, and the [Ca
2
þ
] of the filling solution
should also be in the range of the measured values. With measurements of low
[Ca
2
þ
], Ca
2
þ
in the filling solution can be bu
ered with EGTA to the desired free
[Ca
2
þ
]. However, we have not obtained good results with filling solutions with a
pCa higher than 7.5. We typically use a Ca
2
þ
-EGTA bu
V
er with 1
m
M free Ca
2
þ
as
V
a filling solution.
2. Electrode Characteristics
The resistance of the minielectrodes is 1-2 M
O
, which normally makes it possi-
ble to use a standard pH/ion meter to monitor the electrode potential. We have
used either a commercial pH/ion meter (Orion pH/ion analyzer) or preamplifier
(A311J, Analog Devices). When using commercial pH/ion meters, it is normally
necessary to make an adapter cable that connects the minielectrode to the meter
input. We have used a chart recorder (Soltec) or an electronic data acquisition
device (Linseis) for continuous monitoring of the electrode potential.
The lifetime of the minielectrode depends on the [Ca
2
þ
] the electrode is used to
measure, and on the composition of the experimental solutions. For measurements
in solutions without protein or interfering ions, the detection limit for the electro-
des increases slowly with time and the electrodes will have a detection limit in the
subnanomolar range and a Nernstian response down to 10 nM for at least 1 month
after preparation.
Figure 2
shows the change in the response of a Ca
2
þ
electrode
with time.
Two days after filling, the electrode response was still ''super-Nernstian'' at low
[Ca
2
þ
] (i.e.,
29 mV per 10-fold change in [Ca
2
þ
]), but normal within 7 days after
filling.Notice thatwe did still obtainaNernstian response down topCa 8 for 2months
after filling the electrode. The response time at low [Ca
2
þ
], however, got slower with
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