Life Sciences 67 (2000) 2897–2911

Minireview

Radioligand saturation binding experiments over large

concentration ranges
David B. Bylund*, L. Charles Murrin
Department of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center,
Omaha, NE 68198-6260, USA
Received 10 February 2000; accepted 24 April 2000

Abstract
Receptor analysis using the radioligand binding saturation method in situations requiring a large
concentration range of the ligand is theoretically straightforward but in practice can be relatively diffi-
cult. In this paper we review three approaches for carrying out such experiments and assess the strengths
and weaknesses of each. The three are two saturation experiments, two homologous competition ex-
periments, and the mixed homologous saturation experiment. The best approach depends upon the
experimental conditions and constraints of the system studied, but in general the mixed homologous
saturation experiment appears to be the most effective at minimizing the amount of radioligand needed
while providing the greatest reliability in the results. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Receptor binding analysis; Radioligand binding; Receptor assays; Saturation assays

Introduction
The general guideline for saturation experiments of radioligand binding to receptors is that
a 100-fold concentration range of radioligand should be used (from 10-fold below to 10-fold
above the expected Kd) if the system conforms to a simple one-site model. Although this
range can frequently be achieved using only radioligand (“hot only” experiment), in some
cases this approach is difficult due to poor availability, high cost or low affinity of the radioli-
gand. In addition, frequently it is desirable to use a concentration range greater than 100-fold
in order, for example, to study a more complex binding system. In these cases, the direct “hot
only” approach may not be viable.
A common alternative to the “hot only” approach is the homologous competition experi-
ment in which the concentration of the radioligand is kept constant, and various concentra-
tions of a non-radioactive ligand (with the identical affinity to the radioligand) are used. This
* Corresponding author. Tel.: 402/559-4788; fax: 402/559-7495.
E-mail address: dbylund@unmc.edu (D.B. Bylund)

0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 7 7 -8
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is sometimes called a “cold saturation” experiment and is equivalent to the “hot only” exper-
iment except that the specific activity of the radioligand is different at each radioligand con-
centration. Because the homologous competition experiment does not uniquely define that
portion of the saturation curve at concentrations lower than the lowest concentration of radio-
ligand, the concentration of radioligand used needs to be well below the Kd value. If the spe-
cific activity of the radioligand is not sufficiently high, the specific binding may be too low to
measure accurately.
To overcome the limitations of these two traditional approaches, three additional ap-
proaches are available:
1. Two saturation experiments, the first with radioligand only and a second with similar
radioligand concentrations in the presence of a fixed concentration of the unlabeled
ligand. The addition of the unlabeled ligand should reduce the specific activity of the
radioligand by a factor of five to ten.
2. Two homologous competition experiments, one at a low, and the second at a high, radi-
oligand concentration.
3. A combination of the “hot only” saturation and the homologous competition ap-
proaches, the so-called “mixed homologous saturation” assay.
Although the theoretical aspects of these approaches have been well described [1–6], the
procedures for actually carrying out the assays and analyzing the data have not been given
much attention. For example, a recent paper describing a mixed homologous saturation type
of experiment observed that the “only problem with this type of protocol is that the estimated
Kd could be affected by the differences in affinities between the labeled and unlabeled
ligands” [7]. Our experience with this approach indicates that this problem is not the only one.
Thus, in this report, we review some of the practical aspects of using these three approaches.

Experimental system
For the purposes of illustration, consider an experimental system with the following param-
eters, which are typical of many G protein-coupled receptors: a receptor (alpha-2 adrenergic)
with two subtypes (or affinity states) with affinities of 0.5 nM and 5 nM in equal densities
(40 pM); a radioligand ([3H]RX821002) with a specific activity of 60 Ci/mmol; an assay vol-
ume of 1 ml; and a counting efficiency of 36%.
Fig. 1A presents a typical six-point saturation experiment using this system. The highest
radioligand concentration is 4 nM (200,000 cpm per assay tube). The lowest radioligand con-
centration is 0.05 nM (2500 cpm per assay tube) which results in 178 cpm of specific bind-
ing. Since the Kd of the lower affinity site (5 nM) is higher than the highest radioligand con-
centration (4 nM), the normal saturation assay does not detect the lower affinity site and
nonlinear regression analysis (using the Prism program from GraphPad) gives a single-site
Kd of 0.86 nM and a linear Rosenthal plot (Fig. 1A, insert). The Bmax derived from this exper-
iment is 64 pM which is higher than the Bmax at either site (40 pM), but lower than the total
Bmax for the two sites (80 pM). Similarly, a single homologous competition experiment only
detects one of the sites (Fig. 1B). Using a [3H]RX821002 concentration of 0.4 nM, a single
site is detected (nH 5 0.93) in a typical competition assay with an IC50 of 1.12 nM, which
 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911 2899

Fig. 1. Typical saturation and competition experiments. These theoretical data were derived based on a receptor
population with two affinities (0.5 nM and 5 nM) in equal densities (40 pM) and a radioligand ([3H]RX821002)
with a specific activity of 60 Ci/mmol. Panel A: A typical saturation experiment with free [3H]RX821002 concen-
trations ranging from 0.05 nM (2190 cpm in a 1 ml assay) to 4.02 nM (191429 cpm) and bound [3H]RX821002
concentrations ranging from 3.7 pM (178 cpm) to 53 pM (2543 cpm). Analysis of the data by nonlinear regression
indicates the presence of a single site with a Kd of 0.86 nM and a Bmax of 64 pM. The inset is the Rosenthal [10]
transformation of the data. Panel B: A typical competition experiment with [3H]RX821002 concentration of 0.4 nM
(19,048 cpm). Analysis of the data by nonlinear regression indicates the presence of a single site (nH 5 0.93) with
an IC50 of 1.12 nM corresponding to a Kd of 0.72 nM. In the inset the data are plotted as Bound vs Bound 3
Inhibitor concentration [1]. Similar to the Rosenthal plot, the affinity is the negative reciprocal of the slope.

corresponds to a Kd of 0.72 nM (Kd 5 IC50 2 [radioligand] 5 1.12–0.4; this equation as-

sumes that the receptor concentration is low so that less than 10% of the radioligand is
bound). The Bmax derived from this inhibition experiment is 60 pM (calculated as Bmax 5 Bo
(IC50 /[radioligand]). If a 10-fold higher radioligand concentration is used, a single site inhibi-
tion curve still results (nH 5 0.98) with an IC50 of 5.6 nM, which corresponds to a Kd of
1.6 nM (data not shown). It has been previously pointed out that a single homologous compe-
tition assay generally does not have the power to detect the presence of two classes of bind-
ing sites [8].
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Thus neither of the usual experiments indicates the presence of two sites and both experi-
ments give erroneous values for Kd and Bmax for this system. In the paragraphs that follow,
each of the three above mentioned alternate approaches for extending the concentration range
of the assay will be assessed using this experimental system.

Description of three methods

Two saturation experiments (method one)
Perhaps the most straight forward of the three approaches mentioned above is to perform
two saturation experiments with a 5- to 10-fold difference in specific activity of the radioli-
gand. The first saturation experiment is done in the standard way. For the second experiment,
the radioligand is diluted with non-radioactive ligand (having the identical affinity for the re-
ceptor), which lowers the specificity activity and thus gives higher concentrations without
using higher concentrations of the radioligand. The limiting factor on the amount of unla-
beled ligand that can be added is the number of cpm that will be bound at the lowest concen-
tration of the radioligand. Thus this approach may not be viable in situations where the initial
specific activity of the radioligand is too low or the receptor concentration is so low that a
significant dilution is not possible.

Example of experimental protocol

In this example (Table 1), a 10-fold dilution of the [3H]RX821002 was chosen (to give a
specific activity of 6 Ci/mmol). The two saturation curves with a total of 11 data points span
approximately a 2,000-fold concentration range (0.02 to 41 nM), extending from more than
10-fold below the lower Kd to about 10-fold above the higher Kd.

Table 1
Saturation binding experiment with two different specific activities
Free Added [3H]RX Actual Calculated
[3H]RX [3H]RX Specific act. [3H]RX bound [3H]RX bound
(nM) (cpm) (Ci/mmol) (cpm) (pM)
0.02 1033 60 81 1.7
0.05 2368 60 178 3.7
0.11 5623 60 384 8.1
0.29 14712 60 807 17
0.66 32637 60 1304 27
0.48 2368 6 110 23
1.15 5623 6 169 36
3.06 14712 6 236 50
6.82 32637 6 287 60
17.6 83868 6 333 70
40.7 193972 6 358 75
These data were derived based on a receptor population with two affinities (0.5 nM and 5 nM) in equal densities
(40 pM) and a radioligand ([3H]RX821002; [3H]RX) with an initial specific activity of 60 Ci/mmol and a diluted
specific activity of 6 Ci/mol. These data are illustrated in Fig. 2.
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Note that there is a one point overlap between the two experiments (i.e., the highest con-
centration of the higher specific activity experiment is higher than the lowest concentration of
the lower specific activity experiment). The specific binding (cpm) is quite low in the half of
the experiment done at the lower specific activity. If the cpm were too low to give reliable
data, then a 5-fold rather than a 10-fold dilution could have been used.
By using the specific activity to convert the data to picomolar units of ligand bound, all of
the data can be plotted on the same graph, as is illustrated in Fig. 2. The panels in the left half
of this figure give the data in the standard saturation curve format and as Rosenthal plots in
the insets. The right hand panels give the data with the [3H]RX821002 concentration on a log
scale. With the data combined from the two experiments, a two-site analysis gives the correct
values. The presence of two sites is easily observable, especially in the Rosenthal plot (Fig. 2,

Fig. 2. Saturation experiments with a 10-fold difference in the specific activity of the radioligand. These theoreti-
cal data are from Table 1, and were derived based on a receptor population with two affinities (0.5 nM and 5 nM)
in equal densities (40 pM) and a radioligand ([3H]RX821002) with a specific activity of either 60 Ci/mmol (Higher
Specific Activity) or 6 Ci/mmol (Lower Specific Activity). The left panels give the data in the standard saturation
curve format and as Rosenthal plots [10] in the insets. The right hand panels give the data with the [3H]RX821002
concentration on a log scale. The top panels present the data from the experiment with [3H]RX821002 at a specific
activity of 60 Ci/mmol (open circles). In the middle panels, the specific activity of [3H]RX821002 is 6 Ci/mol
(filled circles) and in the bottom panels, the data from the two experiments are combined.
2902 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911

All Data, inset). In this inset, the two straight lines represent the two receptor populations
with equal densities (40 pM) and a 10-fold affinity difference (slope of the lines).
The LIGAND program ([6]; KELL, Biosoft) has the capability of fitting multiple data sets
at the same time. This program gave correct fits of the data either as separate data sets fit si-
multaneously or as the combined data set (data not shown).

Two homologous competition experiments (method two)

In this experimental approach, a homologous competition curve at a low radioligand con-
centration is used to define the lower part of the saturation curve, whereas a homologous
competition curve with a high radioligand concentration is used to define the higher part of the
curve. The use of a homologous competition method assumes that the reported specific activ-
ity of the radioligand is accurate. An error in this parameter will lead to errors in the Ki values.

Limitations of single competition curves

The higher part of the saturation curve cannot easily be defined using the lower radioli-
gand concentration because the specific binding becomes too close to the nonspecific binding
at the higher concentrations of unlabeled ligand. As the amount of cold ligand is increased,
the specific binding decreases, and eventually approaches zero (total binding approaches
nonspecific binding). Thus, at the highest concentrations, the data become unreliable, because
the specific binding is only a few cpm above nonspecific binding. Looked at another way, the
specific activity of the radioligand becomes very low. If each sample could be counted for
several days, then more accurate data could be obtained, but still the problem of the poor
signal-to-noise ratio over nonspecific binding would remain.
Conversely, the higher radioligand concentration cannot be used to accurately define the
lower part of the competition curve, because data will be obtained only for concentrations at
or above the concentration of radioligand, and thus the estimation of a Kd lower than the radi-
oligand concentration becomes unreliable. Since Kd is the difference between the IC50 and
the concentration of the radioligand (L*), if L* is nearly equal to the experimentally deter-
mined IC50, the Kd becomes the difference between two numbers of similar magnitude, and
thus is not accurate. This is illustrated in Fig. 3, in which the Kd 5 10 nM and L* is varied
from 1 to 100 nM. In this example, when L* is 1nM, the IC50 will be 11 nM and the Kd (11–1)
is 10 nM. By contrast, when L* is 100 nM, the IC50 will be 110 nM and Kd (110–100) is still
10 nM, but the difference between the two numbers (10%) is less than the usual error in the
determination of the IC50 (15–20%). Thus, the data from a homologous inhibition experiment
in which the radioligand concentration is greater than the Kd cannot provide a reliable esti-
mate of the Kd even though the data may appear to be valid.

Example of experimental protocol

Table 2 and Fig. 4 present data using the two homologous competition curves approach for
our experimental system with two populations of receptors (affinities of 0.5 nM and 5 nM in
equal densities). In the top panel of Fig. 4 the radioligand concentration is 0.2 nM and in the
bottom panel it is 2.0 nM. Analysis of the data at the lower radioligand concentration (Ho-
mologous competitive binding curve—One class of binding sites; Prism 3 program) gives a
Kd of 0.65 nM. The B vs B 3 I plot (Fig. 4, upper inset) appears linear (within normal experi-
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Fig. 3. Homologous inhibition curves for different concentrations of radioligand. The curves are simulated based
on a Kd of 10 nM with concentrations of radioligand as noted on the graph. The upper panel presents the data in
cpm and the lower panel presents the data as percent specific binding. The IC50 values for the various curves from
1 nM to 100 nM are 11, 13, 20, 40 and 110 nM respectively.

mental error) and did not indicate the presence of two sites. Attempts to fit the data to a two-
site model were unsuccessful. Similarly, the data in the bottom panel of Fig. 4 (radioligand
concentration of 2.0 nM) appear to represent a single site (Fig. 4, lower inset) and a one-site
fit gives a Kd of 1.33 nM. Attempts to fit these data to a model with two classes of binding
sites were also unsuccessful. The fact that the two concentrations of radioligand give differ-
ent estimates of the affinity would be an indication that the experimental system did not con-
form to a single site model, and the presence of two classes of binding site should be consid-
ered. It should be emphasized, however, that the Kd estimates for the two sites are not
reliable. The “experimentally” determined Kd values were 0.65 and 1.33 nM as compared to
the actual Kd values of 0.5 and 5 nM. For this situation, one approach that has been suggested
is to fit the data from the higher radioligand concentration experiment to a two-site model by
setting the Kd and Bmax for the high affinity site to be constants as determined by the single
site fit of the data from an experiment with a very low radioligand concentration [9]. This
suggestion did not work well for the current data, as the low affinity Kd estimate using this
approach is 29 nM, which is quite different from the actual value of 5 nM. The reason for the
poor fit is that 0.2 nM is not a “very low concentration.” When data generated using a radioli-
2904 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911

Table 2
Homologous inhibition experiments at two different concentrations of radioligand
Radioactive Calculated
Non-radioactive ligand Total Bound bound,
ligand concentration ligand cpm pM
0 0.2 0.2 617 13
0.1 0.2 0.3 548 17
0.3 0.2 0.5 450 24
1 0.2 1.2 286 36
3 0.2 3.2 149 50
10 0.2 10 61 65
30 0.2 30 23 74
100 0.2 100 7 78
0 2 2 2068 43
0.1 2 2.1 2002 44
0.3 2 2.3 1882 45
1 2 3 1565 49
3 2 5 1074 56
10 2 12 529 67
30 2 32 220 74
100 2 102 73 78
300 2 302 25 79
1000 2 1002 8 80
These data were derived based on a receptor population with two affinities (0.5 nM and 5 nM) in equal densi-
ties (40 pM). The final column gives the calculated bound based on the calculated specific activity, which in turn
was calculated from the total ligand concentration and the specific activity of the radioligand (60 Ci/mmol).
Graphs of these data are presented in Figs. 4 and 5.

gand concentration of 0.05 nM were used (excluding unlabeled ligand concentrations which
gave specific binding of less than 30 cpm), then reasonable Kd estimates of 0.53 and 7.9 nM
were obtained (data not shown).
Two other approaches were used in an attempt to find a more reliable method to analyze
the data for the two inhibition experiments. In the first approach, the data were converted
from an inhibition curve to a saturation curve by calculating the bound RX821002 in pM
based on the specific activity of the [3H]RX821002 at each concentration of unlabeled
RX821002 (Table 2). These data are plotted as a saturation curve in Fig. 5. Fitting these data
to a two-site model saturation curve using Prism 3 gives reasonable Bmax and Kd values. The
reason that the same data could not be fit by the analysis as two inhibition curves but could be
fit by the analysis as a single saturation curve, is that in the first analysis the two data sets are
fit separately, whereas in the second all the data are fit at the same time, providing more
power for the analysis.
The LIGAND program fit of the data from the two homologous competition assays gave the
correct results similar to those obtained with the Prism program with the data converted to a
single saturation curve (with the least reliable points removed in both cases). The advantage of
the LIGAND program is that the data do not need to be transformed. The transformation of the
data frequently leads to magnification of relative error associated with some of the data. Also
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Fig. 4. Homologous inhibition experiments at two different concentrations of [3H]RX821002. These theoretical
data were derived based on a receptor population with two affinities (0.5 nM and 5 nM) in equal densities (40 pM)
and a radioligand ([3H]RX821002) at a concentration of either 0.2 nM (upper panel; this concentration is 2-fold
lower than that illustrated in Fig. 1B) or 2.0 nM (lower panel) with a specific activity of 60 Ci/mmol. The insets
present the data as Bound vs Bound 3 Inhibitor [1]. A single-site fit to the data in the upper panel gave a Kd of
0.65 nM and Bmax of 54 pM, whereas a single-site fit to the data in the lower panel gave Kd of 1.33 nM and Bmax
of 72 pM. When the data in the lower panel were fit to a two-site model with the Kd and Bmax of the high affinity
site held constant at the values derived from the fit in the upper panel, the derived Kd for the low affinity site was
29 nM and the Bmax was 47 pM.

the full data set can be used in the LIGAND program, thus avoiding decisions about which
data points are unreliable. It should be noted however, that the results were closer to the correct
values when the data points with the lowest cpm were excluded. The disadvantage with using
LIGAND is that the program is significantly more difficult to use. The graphic capabilities of
the LIGAND program are very limited, whereas the graphic capabilities of Prism are excellent.
Mixed homologous saturation (method three)
The mixed homologous saturation assay is a combination of a homologous competition
assay and a “hot only” saturation assay. A typical saturation curve is used to define the lower
2906 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911

Fig. 5. Competition data converted to saturation data. The data from Fig. 4 were replotted as a saturation curve by
calculating the new specific activity of the [3H]RX821002 at each concentration of unlabeled RX821002 (Table 2).
The data for the highest two concentrations of unlabeled RX821002 at each of the radioligand concentrations are
not included in these data because the cpm (23 and 7 for 0.2 nM, and 25 and 8 for 2 nM) were too low to be reli-
able. A change of only 1–2 cpm in actual binding leads to large changes of 7–25 pM in the converted data. A fit of
these data to a two-site model gave the correct Kd values of 0.5 and 5 nM, and Bmax values of 40 pM.

part of the saturation curve starting with the lowest radioligand concentration that gives reli-
able data. This concentration should be at least 10-fold lower than the high affinity Kd value.
A homologous competition assay is used to define the upper part of the saturation curve, re-
ducing the amount of radioligand needed. The radioligand concentration used for this part of
the experiment should be lower than the highest concentration of the saturation experiment
so that there is overlap in the data produced by the two parts of the experiment.
Example of experimental protocol
The results of the mixed homologous saturation data for our experimental system are pre-
sented in Fig. 6A. As expected, the analysis of these data also gives the correct results. The
advantage of the mixed homologous saturation approach (Method Three) is that the lowest
part of the curve is better defined than with Method Two (Two Competition Experiments)
and the upper part of the curve is better defined than with Method One (Two Saturation Ex-
periments). Significantly less radioligand is used in Methods Two and Three as compared to
Method One. This is a marked advantage in some situations such as in autoradiographic ex-
periments which use large incubation volumes.
Similar results were obtained using both the Prism and LIGAND programs. The advan-
tage of the LIGAND program is that a saturation and inhibition curve can be fit simulta-
neously without converting the homologous competition data to saturation data. An alternate
way of presenting data from a mixed homologous experiment is to plot Bound/Total vs Total
ligand as is shown in Fig. 6B [7]. However, presenting the data as a saturation curve (Fig.
6A) appears to be preferable since the y-axis is more familiar and the value of Bmax can be di-
rectly estimated. We see no advantage to the Bound/Total vs Total plot.
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Fig. 6. Mixed homologous saturation assay which combines a homologous competition and a saturation assay.
These theoretical data were derived based on a receptor population with two affinities (0.5 nM and 5 nM) in equal
densities (40 pM). Panel A. The data for the lower part of the curve (open circles) are from the saturation data
given in the top half of Table 1 (specific activity of 60 Ci/mmol) and the upper panel of Fig. 2 (Higher Specific
Activity Data). The data for the upper part of the curve (closed squares) are from the homologous competition
data given in the bottom half of Table 2 (radioactive ligand concentration of 2.0 nM; converted to saturation data)
and in Fig. 5 (closed squares). Panel B. The same data as in Panel A plotted Bound/Total vs Total ligand.

Method three with real experimental data

Data from an actual mixed homologous assay are presented in Table 3. This assay was
done with 16 mm sections of rat brain on glass microscope slides as would be done for an au-
toradiographic experiment. At the end of the experiment the bound radioactivity in the tissue
was determined. The first half of the experiment is a standard saturation experiment with ra-
dioligand concentrations from 0.013 nM to 0.78 nM. The second half of the experiment is a
homologous competition experiment using 0.165 nM radioligand and concentrations of unla-
beled ligand from 0.1 to 300 nM. The homologous competition data are transformed to calcu-
lated bound based on the calculated specific activity of the radioligand at each concentration
of unlabeled ligand, and are graphed in Fig. 7. Note that by using this mixed homologous sat-
uration method, the useful concentration range of the radioligand is from 0.013 to 30 nM,
which is a 2300-fold range.
2908 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911

Table 3
Example of an actual mixed homologous assay
Specific Calculated
Non-radioactive Radioactive Total binding, bound
ligand ligand ligand dpm dpm
0 0.013 0.013 27 27
0 0.019 0.019 41 41
0 0.042 0.042 110 110
0 0.076 0.076 203 203
0 0.156 0.156 390 390
0 0.406 0.41 814 814
0 0.772 0.77 1077 1077
0.1 0.165 0.27 352 565
0.3 0.165 0.47 333 938
1 0.165 1.17 192 1358
3 0.165 3.2 94 1809
10 0.165 10.2 27 1684
30 0.165 30 9 1584
100 0.165 100 1 405
300 0.165 300 1 1819
Sections (16 mM) of rat brain were incubated with the indicated amount of labeled and unlabeled RX821002
for 4 hr at room temperature on glass slides. Nonspecific binding was determined with 10 mM rauwolscine. The
sections were washed twice for 5 min in ice-cold 50 mM Tris-Mg-EDTA buffer and wiped from the slide with a
glass fiber filter and the radioactivity determined. These data are presented graphically in Fig. 7.

The final two data points were not included in the fit of the data (although they are on the
graph as open squares) because they each were only one dpm over background. This empha-
sizes a major limitation of homologous competition experiments. There are several ways to
increase the specific binding at the high concentrations of unlabeled ligand. One is to use a
higher concentration of radioligand. In these experiment this was not practical because the
incubation volume for the slides was 9 ml and thus increasing the concentration would be too
costly. (An alternate autoradiographic procedure in which a small volume of incubation
buffer placed directly on the slide-mounted tissue section using a hydrophobic barrier, would
allow for higher concentrations of the radioligand at a reasonable cost. With this approach,
however, buffer evaporation can be a significant problem). The second way would be to use
more tissue, which is similarly not practical in the current experiments.
Using Prism, there are three options for weighting the data in curve fitting: no weighting,
which assumes equal absolute error for all data points; 1/Y2, which assumes equal relative er-
ror (essentially the error divided by the value); and 1/Y which is a compromise between the
other two. For homologous competition the 1/Y2 weighting is best since the magnitude of the
error in the calculated bound increases as the value increases. However, a certain amount of
judgement must be used, because if there is considerable scatter in the data, the results could
be misleading. For example, if all of the data are used from the actual mixed homologous sat-
uration experiment (Table 3), the 1/Y2 weighting does not give a reasonable result. The upper
panel of Fig. 8 presents the results using three different weighting options: no weighting;
1/Y weighting; and 1/Y2 weighting. Using the 1/Y2 weighting, the data point at 1027 M
 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911 2909

Fig. 7. Example of an actual mixed homologous saturation assay. Sections (16 mm) of rat brain were incubated
with various amounts of labeled and unlabeled RX821002 for 4 hr at room temperature on glass slides. The sec-
tions were washed twice in buffer, wiped from the slide with a glass fiber filter and the radioactivity determined.
The data are given in Table 3. The open circles are the data from the saturation assay and the squares are from the
homologous competition assay. The data points represented by the open squares were not included in the analysis,
because they each were only one dpm over background. The solid line is the one-site fit of the data with1/Y2
weighting and the slope variable (Kd 5 0.45 nM, Bmax 5 1740 dpm/section, nH 5 1.14) . The dashed line is the
one-site fit of the data with1/Y2 weighting and the slope fixed at 1.0 (Kd 5 0.55 nM, Bmax 5 1844 dpm/section).
The fit with the variable slope was significantly better (F(1,9) 5 6.7, p 5 0.03) than the fit with the slope fixed at 1.0.

RX821002 has a large effect on the analysis (because the value is so low), whereas the effect
of this point is much less when no weighting is used. In the lower panel, the data point at
1027 M RX821002 was switched to as far above the expected value as it was below the ex-
pected value in the upper panel. In this case the 1/Y2 weighting gives the most reasonable
analysis.
The two data sets (competition and saturation) from the actual experiment were also ana-
lyzed simultaneously with the LIGAND program and similar results to those from Prism
were obtained (Kd 5 0.65 nM). In our hands the optimum weighting method using LIGAND
had to be determined by trial and error. In the analyses for the data in this paper, a weighting
of 1/Y2 usually gave results closest to the expected values. In at least one case, however, no
weighting provided the best fit. The reason for this difference was not apparent.

Conclusions
Saturation radioligand binding experiments are a powerful tool in the study of many re-
ceptors. There are several experimental situations in which it is useful or necessary to use a
2910 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911

Fig. 8. Effect of weighting in the analysis of mixed homologous saturation data. The data are taken from Table 3
and Fig. 7. The upper panel presents the results of fitting all of the data to a single site sigmoidal model using the
three different options to weight the data which are available in the Prism 3 program: no weighting; 1/Y weight-
ing; and 1/Y2 weighting. The lower panel is identical except for the data point at 1027 M RX821002, which was
switched to as far above the expected value as it was below the expected value in the upper panel. For all analyses
the fit of the data to the variable slope model was not significantly better than the fit with the slope fixed at 1.0.
The faint solid curve in both panels is the solid curve from Fig. 7.

ligand concentration range greater than the traditional 100-fold range. In some of these situa-
tions, it is not practical to use only the radioligand. Three experimental approaches to deal
with this situation were assessed in this article: two saturation experiments; two homologous
competition experiments; and the mixed homologous saturation experiment. Whereas all
three approaches can produce reliable results if the data are analyzed correctly, the mixed ho-
mologous saturation experiment appears to have the advantage because it can best define
both the lower and upper parts of the saturation curve with the use of the minimum amount
of radioligand.

Acknowledgments
The authors gratefully acknowledge the support of NIH grant NS33194.
 D.B. Bylund, L.C. Murrin / Life Sciences 67 (2000) 2897–2911 2911

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