A Practical Guide to the Preparation of Ca Buffers - Methods in Cell Biology - page 13

chemicals, etc.). Thus, it is valuable to measure the free [Ca 2 þ ] to check that the
solutions are as you expected (especially for complex solutions). Ca 2 þ sensitive
electrodes are a convenient way to do this (see Chapter 3). We normally use
Ca 2 þ minielectrodes (as described in Chapter 3) or commercial macroelectrodes.
Both can be connected to a standard pH meter, but it is best to have a meter
which can read in increments of 0.1 mV. We have had good luck with Orion brand
Ca 2 þ -electrodes and they can be stable for 6 months or so. However, they are
rarely as good as the home-made minielectrodes. These minielectrodes are very
easy to make and are sensitive to changes in free [Ca 2 þ ] down to 1 nM or beyond.
They do not last as long as commercial macroelectrodes, but they are extremely
cheap to make (per electrode) and can be discarded if they get contaminated with
protein or are exposed to radioactive molecules. One can also use fluorescent
indicators, once suitably calibrated, in an analogous way. The only disadvantage
there is the more limited dynamic range of these Ca 2 þ indicators (10-fold
above and below the K d ) versus electrodes which can give linear responses over
the 10 nM-1 M range.

2. Spreadsheet for Calibration Calculations
Making up calibration solutions for Ca 2 þ -electrodes (or fluorescent indicators)
is really a simpler version of the multiple equilibria problem which will be discussed
below (with respect to MaxChelator), because we really only need to consider the
Ca 2 þ -EGTA bu

er system. This approach is based on the paper by Bers (1982) .
This method has the following general steps:
1. Calculate how much total Ca 2 þ (or free [Ca 2 þ ]) is required for the desired
solutions (using known constants, corrected as above). All solutions should have
the same dominant ionic constituents as the solutions to be measured (e.g.,
140 mM KCl, 10 mM HEPES).
2. Measure the free [Ca 2 þ ] with a good quality Ca 2 þ electrode compared to free
[Ca 2 þ ] standards without EGTA (at higher [Ca 2 þ ] where [Ca 2 þ ] is more easily
controlled).
3. Accepting (for the moment) that the values from the electrode are all correct,
allows the calculation of bound Ca 2 þ ([CaR]) from free [Ca 2 þ ] and total [Ca 2 þ ].
4. Scatchard plot analysis allows the independent measurement of the apparent
K 0 Ca and total [EGTA] in your solutions and experimental conditions (even with
systematic errors). Note that the Scatchard plot is very sensitive and deviates from
linearity at very low [Ca 2 þ ] where Ca 2 þ -electrodes can become sub-Nernstian in
response (see Figs. 6 and 7 ).
5. Using these ''updated'' values of total [EGTA] and K 0 Ca you can recalculate
the free [Ca 2 þ ] in the solutions. Then you can either use the free [Ca 2 þ ] predicted
from the electrode directly or you can recalculate from the total [Ca 2 þ ] and

V