Biology Reference
In-Depth Information
B
A
40,000
0
Original pCa
Electrode pCa
Recalc. pCa
Regression line
30,000
−50
20,000
−100
10,000
−150
0
0
1
2
3
4
5
3
4
5
6
7
89
Bound Ca
2+
-EGTA (mM)
pCa
Fig. 7
Scatchard plot (A) and electrode calibration curves (B) for the spreadsheet shown in
Fig. 6
. The
Scatchard plot allows estimation of the total [EGTA] (x-intercept) and the apparent association
constant, K
0
Ca
(-slope). The Scatchard plot is very sensitive to the detection limit of the Ca
2
þ
electrode.
The leftmost two points in A are the lowest free [Ca
2
þ
] in the calibration curve in B (and are not included
in the regression). The three calibration curves shown are for the original (or planned pCa), the pCa
predicted solely by the electrode and the pCa after recalculation, using the values determined in the
Scatchard plot along with the total Ca
2
þ
added to the bu
V
ers. In this instance, there was good agreement
between the three curves, but this is not always the case (see
Bers, 1982
).
updated constants. The latter is necessary for the lowest free [Ca
2
þ
] where the
electrode response is becoming nonlinear (
pCa 9).
We use a spreadsheet (Excel) to greatly simplify all of these steps (see
Fig. 6
).
There are three basic versions of this spreadsheet: one for starting with free
[Ca
2
þ
] as the input (DMB-CAF-2010.xls), one for pCa as input (DMB-PCA-
2010.xls), and one for total Ca
2
þ
as input (DMB-CAT-2010.xls). These can be
freely downloaded from the MaxChelator site as described below. We will walk
you through the use of this spreadsheet in making a series of free [Ca
2
þ
]
standards here.
The fields for the input of data are shaded dark gray. For the pCa version of the
spreadsheet in
Fig. 6
you proceed as follows (the others versions are completely
analogous):
1. Enter your solution conditions (upper left, pH, ionic strength, temperature,
total [EGTA], and bottle size you will use). The K
0
Ca
values are then automatically
adjusted for the selected temperature, pH, and ionic strength (lower left box).
2. Enter the desired pCa values. The free [Ca
2
þ
], total [Ca
2
þ
], and ml of 100 mM
Ca
2
þ
stock are automatically calculated (next three columns) using the adjusted
K
0
Ca
.
