A Practical Guide to the Preparation of Ca Buffers - Methods in Cell Biology - page 16

3. Enter the mV readings from a Ca 2 þ electrode (including values for Ca 2 þ
standards lacking EGTA at 100 m M, 1 mM, and 10 mM [Ca 2 þ ] and the electrode
reading at 1 mM free [Ca 2 þ ] as the ''o

set''). This is the fourth column (V-Ca) and
you can choose the electrode slope (rather than assume the average). The free
[Ca 2 þ ], Ca 2 þ -bound to EGTA, and the bound/free (B/F) are then automatically
calculated (based on the electrode response and total [Ca 2 þ ]).
4. Those calculated values (light shaded box, yellow in downloaded file) will be
subject to linear regression Scatchard analysis (automatically). The Scatchard plot
and Electrode calibration curves ( Fig. 6 , bottom) can be inspected to check
linearity. If values within the regression window are not on the linear range, they
can throw o

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the analysis. The top and bottom two [Ca 2 þ ] are excluded from the
regression to allow calculations of [Ca 2 þ ] for solutions outside that range.
5. Finally, the free [Ca 2 þ ] and pCa are automatically recalculated using the
measured K 0 Ca and total [EGTA] (from the auto-analysis) as well as the total
Ca 2 þ values (last two columns). The Electrode calibration curve and Scatchard
plot allow you to get an overview of the results. ( Fig. 6 ).
We routinely use this for calibration solutions for both Ca 2 þ -electrodes and
fluorescent indicators. In addition to improving the reliability of Ca 2 þ calibration
solutions, one of the convenient aspects of this spreadsheet is that you can see all
the details of what is going on. For example, you can see that the EGTA is almost
completely saturated as you get up to 10 m M free [Ca 2 þ ]. In this range we usually
believe the electrode, rather than our ability to pipette within 1% of the required
volume. On the other hand, as you approach the detection limit of the electrode
(e.g.,

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pCa 9), we use the recalculated pCa values. The measured versus predicted
K 0 Ca , EGTA purity and [Ca 2 þ ] can also be useful in identifying potential systematic
errors or changes in your procedures.

B. Preparing Bu er Solution

1. Basic Steps in Solution Preparation
There are no hard and fast rules or special tricks to make these bu

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ers, but
special care in weighing and pipetting, and common sense can help avoid some
potential problems. The water should be well purified to minimize contamination
with Ca 2 þ and other metals. We usually use water that is first distilled and then run
through a water purification system containing at least one ion exchange column
(e.g., Nanopure,

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from Barnstead). This provides water with resistivity of

> 15 MOhm-cm. Starting with good water like this is important for removal of

other metal contaminants as well as Ca 2 þ . There can also be contaminating Ca 2 þ
and metals in the salts and chemicals used to make solutions. In the end, it is
typical to find 1-3 m M free [Ca 2 þ ] in nominally Ca 2 þ -free solutions. This can be
checked with a Ca 2 þ -electrode.