Biophys Rep 2015, 1(3):148–155

DOI 10.1007/s41048-016-0016-5 Biophysics Reports

P R OTO C O L

Radioligand saturation binding for quantitative analysis

of ligand-receptor interactions
Chengyan Dong1, Zhaofei Liu2, Fan Wang1,2&
1
Interdisciplinary Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
2
Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic Medical Sciences, Peking
University, Beijing 100191, China

Received: 10 November 2015 / Accepted: 20 December 2015 / Published online: 14 February 2016

Abstract The reversible combination of a ligand with specific sites on the surface of a receptor is one of the most
important processes in biochemistry. A classic equation with a useful simple graphical method was
introduced to obtain the equilibrium constant, Kd, and the maximum density of receptors, Bmax. The
entire 125I-labeled ligand binding experiment includes three parts: the radiolabeling, cell saturation
binding assays and the data analysis. The assay format described here is quick, simple, inexpensive, and
effective, and provides a gold standard for the quantification of ligand-receptor interactions. Although
the binding assays and quantitative analysis have not changed dramatically compared to the original
methods, we integrate all the parts to calculate the parameters in one concise protocol and adjust many
details according to our experience. In every step, several optional methods are provided to accom-
modate different experimental conditions. All these refinements make the whole protocol more
understandable and user-friendly. In general, the experiment takes one person less than 8 h to com-
plete, and the data analysis could be accomplished within 2 h.

Keywords Equilibrium constant, Maximum density of receptors, Saturation binding assays, I-125 labeling,
Radioligand

INTRODUCTION et al. 2012). This radioligand binding assay (RBA)

remains the most sensitive quantitative approach to
Research on receptors has developed very quickly in the measuring binding parameters in vitro, even in low
past few decades. The interactions between ligands and receptor-expression cells (Rovati 1993; Keen 1995).
receptors generate and enhance signals for recognition, Since the 1970s, the application of RBA has developed
feedback and crosstalk in cells (Klotz 1985; Wilkinson rapidly, with better receptor preparations, more radio-
2004). Receptors are divided into two groups by their labeled ligands and higher radioactivity. Currently,
location: in the membrane or the nucleus. Hormones appropriate ligands radiolabeled with tritium or iodine
and transmitters can selectively recognize and bind to are available for the study of many receptors, including
receptors to accomplish a biological process. Many adrenergic, cholinergic, dopaminergic, serotonergic, and
drugs are designed and improved based on utilizing the opiate receptors (Tallarida et al. 1988). This widespread
ligand-receptor interaction. availability has led to a rapid growth in the use of radi-
Radioligand binding is widely used to define receptor oligand binding assays to characterize novel receptors
function at the molecular level. The first radiolabeled and receptor subtypes and determine their anatomical
binding assay was developed during the 1960s (Maguire distribution, and these assays play a vital role in the
development of drugs by the pharmaceutical industry
(Bylund and Toews 1993; Carpenter et al. 2002).
& Correspondence: wangfan@bjmu.edu.cn (F. Wang)

148 | December 2015 | Volume 1 | Issue 3  The Author(s) 2016. This article is published with open access at Springerlink.com
Radioligand binding for quantitative analysis of ligand-receptor interactions PROTOCOL

Unlabeled ligands require a radioactive isotope to be For further analysis, [SB] could represent the con-
incorporated into the molecule. It is occasionally nec- centration of ligand bound to the receptor, [F] repre-
essary to modify the structure of the ligands to provide sents the concentration of unbound ligand or ‘‘free’’
a suitable site for radiolabeling. However, it is imper- ligand, and Bmax represents the greatest attainable
ative that the selectivity and specificity of the ligand be concentration of bound ligand:
retained after the modification and radiolabeling. Two
½SB Bmax ½RL
basic parameters of this binding site can be studied by ¼  : ð5Þ
½F Kd Kd
kinetic and saturation analysis: the affinity of the
ligand for its recognition site, the Kd, and an estimate Thus, a plot of fractional [SB]/[F] vs. [RL] will give
of the number of binding sites in a given tissue, the the basic information of the ligand-receptor interac-
Bmax (Williams and Jacobson 1990). The Kd is the tions, known as a ‘‘Scatchard plot’’. An alternative is the
equilibrium dissociation constant, which is the con- Woolf plot, a plot of fractional [F]/[SB] vs. [L]:
centration of ligand that will occupy 50% of the ½F Kd ½L
receptors. The generally accepted standard is that a ¼ þ : ð6Þ
½SB Bmax Bmax
ligand-receptor binding with a Kd of 1 nmol/L or less
has a high affinity, whereas ligands binding with a Kd
of 1 lmol/L or more have low affinity (Davenport and
Russell 1996). Bmax signifies the maximum density of APPLICATIONS AND LIMITATIONS
receptors. This value is unique to a particular tissue in
the binding assay, and it is usually corrected using the Radioligand-binding techniques are applicable to any
amount of protein or cells present. receptor of interest, provided it has a relative ligand that
could selectively bind the receptor and be labeled with
radioactive isotopes. Antibodies, proteins and peptides
MECHANISM OF ACTION that contain Tyr could easily be labeled with iodine.
Radioligands provide precise probes to quantify the
Analysis of radioligand binding experiments is based initial interaction between ligands and receptors. For
on a simple model; the law of mass action that example, the kinetics of the association and dissociation
describes the interaction between one molecule of of radioligands can be accurately examined from a
ligand and one receptor molecule. For example, a simple tissue expressing the specific target receptors. A
neurotransmitter binds to the synaptic receptor to series of concentrations of unlabeled ligands could
initiate the neurobiological process, or an antibody inhibit the forces established between radioligands and
binds to an antigen to initiate the immunological receptors, which could be used to measure the equi-
response. In the simplest and most common case, this librium dissociation constants. Radioligand binding also
is a bimolecular reaction between a ligand and a permits a characterization of receptor subtypes with
receptor. This model assumes that binding is reversible different affinities and provides an estimate of their
(Anderson 1994). relative proportions, especially in the study of central
½R þ ½L  ½RL; ð1Þ nervous system receptors, where the effects of neuro-
transmitters are complex, and isolated tissue prepara-
where [R] is the concentration of free receptor, [L] is the tions are unfeasible (Tallarida et al. 1988). Radioligand
concentration of free ligand, [RL] is the concentration of binding assays can also be used to monitor the changes
the complex, k1 is the association rate constant, and k2 is in receptor density, perhaps resulting from the patho-
the dissociation rate constant, logical conditions of pharmacological intervention.
k2 ½R½L Furthermore, the Scatchard plot is a useful diagnostic
Kd ¼ ¼ : ð2Þ
k1 ½RL tool to determine whether more than one ligand mole-
cules bind to a single receptor (Hollemans and Bertina
The density of unbound receptors [R] and ligands [L] 1975; Rovati 1998). A concave upward plot is indicative
cannot be determined but could be given: of nonspecific binding, negative cooperativity, or multi-
½R ¼ ½RL  ½RL: ð3Þ ple classes of binding sites. A concave downward plot
suggests either positive cooperativity or instability of
Hence, Eq. 2 could be rearranged to: the ligand (Wilkinson 2004).
½RL ½RT ½RL However, binding parameters could be affected by
¼  : ð4Þ many factors including the specific radioactivity, the
½L Kd Kd

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PROTOCOL C. Dong et al.

type and ionic strength of the buffer, the presence of 12 Add 4–7 tubes of standard samples to measure.
divalent ions and the temperature. The results are not Then measure the radioactivity on each mem-
sufficient to reflect the real physiological response brane with a c-counter.
mediated by the receptor in this homogenate prepara- 13 For each experimental measurement, subtract the
tion, such as minimizing degradation of the ligand cpm values of groups. Added activities [TA], total
(Davenport and Russell 1996). In addition, radioligand- binding activities [TB] and non-specific binding
binding assays cannot adequately discriminate between activities [NSB] could be measured and calculated
full agonists that elicit maximal physiological responses by the activities on the membranes of plates.
and partial agonists that cannot elicit a maximal 14 Calculate [SB], [LT], [RL], [L] and [F] using these
response (Tallarida et al. 1988). corrected values.
15 Kd value and Bmax could be calculated by Scatchard
Plot, Woolf Plot or the software.
SUMMARIZED PROCEDURE

1 Dissolve iodogen in chloroform at a concentration

PROCEDURE
of 2 mg/mL, evaporate the chloroform and make
the iodogen-coated tube under an N2 stream.
Radiolabeled protein preparation [TIMING] ~1 h
2 Dissolve Protein L in 0.2 mol/L phosphate buffer
(pH 7.4) at a concentration of *2 mg/mL.
1 There are two options for labeling the proteins.
3 Mix 50 lL Protein L, 50 lL PB (0.2 mol/L, pH 7.4)
and 50–100 lL Na125I ([40 MBq) in an (A) Option A: Chloramine-T method (Hunter
iodegen-coated tube. Incubate for 7–8 min at 1970; Opresko et al. 1980).
room temperature. i. Dissolve Protein L (see ‘‘Reagent setup’’ sec-
4 Remove the reaction mixture from the iodogen tion) in 0.2 mol/L phosphate buffer (pH 7.4)
tube and purify the radiolabeled protein by size at a concentration of 2 mg/mL.
exclusion chromatography using a PD MidiTrap ii. Mix the following reagents: 50 lL Protein L,
G-25 column. 50–100 lL Na125I ([40 MBq) and 100 lg
5 Count the activity in the final recovered tube and Chloramine-T (1 mg/mL in 100 lL 0.2 mol/L
calculate the specific activity. PB, pH 7.4). Incubate for 40 s at room
6 Wash two 96-well plates with pre-cooled cell- temperature.
binding buffer for three times (100 lL each well) [CRITICAL STEP] It is highly recommended to
and use the vacuum manifold to remove the buffer. limit the reaction time in 1–3 min.
Place two 96-well plates for specific binding and iii. Add 100 lL Na2S2O5 (200 lg in ddH2O) and
non-specific binding respectively. 100 lL 1% KI (1 mg in ddH2O) to the mixture.
7 From a stock of two million receptor- (B) Option B: Iodogen method (Bailey 1996).
overexpressed cells per mL of cell-binding buffer i. Dissolve Protein L (see ‘‘Reagent setup’’ sec-
(total volume [ 1.5 mL), add 1 9 105 cells tion) in 0.2 mol/L phosphate buffer (pH 7.4)
(50 lL) each well in the 96-well plate. at a concentration of *2 mg/mL.
8 Prepare three stock solutions of different concen- ii. Dissolve iodogen in chloroform at a concen-
tration 125I-Protein L (e.g., 0.1, 1 and 10 lg/mL) in tration of 2 mg/mL. Transfer aliquots of
cell-binding buffer. 25 lL (50 lg) to a glass-bottomed screw cap
9 Add the cells and 125I labeled ligand into the vial. Evaporate the chloroform to dryness
plates according to calculation of final concentra- under an N2 stream, leaving a thin coating of
tion for each well (N = 4), and adjust the total iodogen in the tube. Store the desiccated
volume to 200 lL per well with cell-binding buffer iodogen-coated tubes at -20 C until
and incubate for 2 h at 4 C. required for iodination.
10 Use the vacuum manifold to remove the incuba- iii. Mix 50 lL Protein L, 50 lL PB (0.2 mol/L, pH
tion buffer from the plates and wash 5–10 times 7.4) and 50–100 lL Na125I ([40 MBq) in an
with cell-binding buffer (100 lL/well). iodogen-coated tube. Incubate for 7–8 min at
11 Heat-dry the plates in the dry bath incubator, and room temperature.
collect the membrane from each well into poly- [CRITICAL STEP] It is highly recommended
styrene culture test tubes. to maintain the duration of the reaction
between 5–10 min.

150 | December 2015 | Volume 1 | Issue 3  The Author(s) 2016. This article is published with open access at Springerlink.com
Radioligand binding for quantitative analysis of ligand-receptor interactions PROTOCOL

iv. Remove the reaction mixture from the iodo- Saturation binding [TIMING] 5–7 h
gen tube and apply to purifying.
[CAUTION!] It is imperative to obtain appro- 4 Wash two 96-well plates with pre-cooled cell-
priate training from the institutional radiation binding buffer three times (100 lL each well) and
safety office before experimenting with use the vacuum manifold to remove the buffer.
radioactivity. Abide by all relevant regulatory One 96-well plate (Plate A) will be used for
rules and use appropriate protection when specific binding and the other one (Plate B) will be
handling radioactivity. Dispose of the 125I- used for non-specific binding(Cai and Chen 2008).
containing radioactive waste according to the 5 From a stock of two million receptor-overexpressed
institutional radioactive waste disposal cells per mL of cell-binding buffer (total volume [
guidelines. 1.5 mL), add 1 9 105 cells (50 lL) to each well in
the 96-well plate.
2 Purification: Purify the radiolabeled protein by size
6 Prepare three stock solutions of different concen-
exclusion chromatography using a PD MidiTrap
trations of 125I-Protein L (e.g., 0.1, 1 and 10 lg/mL)
G-25 column.
in cell-binding buffer. Typically a series of concen-
(A) Preparation and equilibration: Remove the trations between 1 ng/200 lL and 1 lg/200 lL
caps and the storage solution. Fill the column will be needed per well.
with PBS and discard the flow-through. [CAUTION!] It is imperative to obtain the appro-
Repeat this procedure twice (three times in priate preparatory training and abide by all regu-
total). latory rules when handling radioactivity.
(B) Sample application: Add a maximum of [? TROUBLESHOOTING]
1.0 mL of sample to the column. Apply the 7 Add the cells and 125I-labeled ligand into Plate A
sample slowly in the middle of the packed bed following Table 1, and adjust the total volume to
and discard the flow through. 200 lL per well with cell-binding buffer and
(C) Elution: Place a clean tube for sample collec- incubate for 2 h at 4 8C. More than four samples
tion under the column. Elute with 1.5 mL PBS are recommended for each concentration.
and collect the products. [? TROUBLESHOOTING]
8 Add the cells, 125I-labeled ligands and excess cold
3 Count the activity in the final recovered tube. The
Protein L into Plate B following Table 2, and adjust
specific activity is calculated as the quotient
the total volume to 200 lL per well with cell-
between the recovered activity and the total amount
binding buffer and incubate for 2 h at 4 8C as the
of protein. This calculation assumes that 100% of
last step. More than four samples are recom-
the protein added to the iodination was recovered,
mended for each concentration.
which is not typical. (Analytical Techniques, T.P.
[? TROUBLESHOOTING]
Mommson)
9 Use the vacuum manifold to remove the incuba-
Radioactivity tion buffer from the 96-well plate and wash 5–10
Specific activity ¼ (Ci/g):
Protein mass times with cell-binding buffer (100 lL per well).
[CRITICAL STEP] To obtain an optimal result, it is 10 Heat-dry the 96-well plates in a dry bath incuba-
sufficient to utilize radioligands with high specific tor until all filter membranes are dry. This usually
activity ([20 Ci/mmol). takes approximately 15 min.

Table 1 Sample adding Specific binding group

strategy in the typical 96-well
plate for the specific binding No. 1 2 3 4 5 6 7 8 9 10 11 12
assay
125
I-Protein L (lg/mL) 0.1 0.1 0.1 0.1 1 1 1 1 10 10 10 10
125
I-Protein L (lL) 10 20 50 80 10 20 50 80 10 20 50 80
Binding buffer (lL) 140 130 100 70 140 130 100 70 140 130 100 70
Cell solution (lL) 50 50 50 50 50 50 50 50 50 50 50 50
Total volume (lL) 200 200 200 200 200 200 200 200 200 200 200 200

 The Author(s) 2016. This article is published with open access at Springerlink.com 151 | December 2015 | Volume 1 | Issue 3
PROTOCOL C. Dong et al.

Table 2 Sample adding Non-specific binding group

strategy in the typical 96-well
plate for the non-specific No. 1 2 3 4 5 6 7 8 9 10 11 12
binding assay
125
I-Protein L (lg/mL) 0.1 0.1 0.1 0.1 1 1 1 1 10 10 10 10
125
I-Protein L (lL) 10 20 50 80 10 20 50 80 10 20 50 80
Cold Protein L (lL) 50 50 50 50 50 50 50 50 50 50 50 50
Binding buffer (lL) 90 80 50 20 90 80 50 20 90 80 50 20
Cell solution (lL) 50 50 50 50 50 50 50 50 50 50 50 50
Total volume (lL) 200 200 200 200 200 200 200 200 200 200 200 200

11 Collect the membrane from each well into poly- (A) Option A: Scatchard plot
styrene culture test tubes. i. For each point on the concentration, enter
12 Add 4–7 tubes of radiolabeled samples (standard [RL] into the X column and the value of
samples) to measure. [SB]/[F] into the Y column:
[PAUSE POINT] The radioactivity on each mem-
brane can be measured later, because 125I has a ½SB Bmax ½RL
half-life of 60 d. ¼  : ð12Þ
½F Kd Kd
13 Measure the radioactivity on each membrane with
a c-counter. ii. All the points are plotted and then linear
regression is used to produce the line.
iii. Referring to the Results sheet for the
Analysis [TIMING] 1–2 h
regression analysis, the X- and Y-axis inter-
cepts could be calculated. The X-intercept
14 For each experimental measurement, subtract the
represents the Bmax, and the Y-intercept
cpm values of the groups. Added activities [TA]
represents Bmax/Kd.
could be measured using the standard samples.
Total binding activities [TB] and non-specific
binding activities [NSB] could be measured by the (B) Option B: Woolf plot
activities on the membranes of Plate A and B, i. For each point on the concentration, enter
respectively. [L] into the X column and the value of [F]/
15 Using Eqs. (5)–(9), calculate [SB], [LT], [RL], [SB] into the Y column:
[L] and [F] using these corrected values:

½F Kd ½L
½SB ¼ ½TB  ½NSB; ð7Þ ¼ þ : ð13Þ
½SB Bmax Bmax
½TAðcpmÞ
½LT ¼ ii. All the points are plotted and then linear
E%  ½SAðlCi=nmolÞ  2:22  106
regression is used to produce the line.
103
 ðpmol=LÞ; ð8Þ iii. Referring to the results sheet for the regres-
Volume ðLÞ
sion analysis, the X- and Y-axis intercepts could
½SBðcpmÞ be calculated. The X-intercept represents the
½RL ¼
E%  ½SAðlCi=nmolÞ  2:22  106 Kd, and the Y-intercept represents Kd/Bmax.
103
 ðpmol=LÞ; ð9Þ
Volume ðLÞ
(C) Option C: Saturation binding curve
½L ¼ ½LT  ½RL; ð10Þ i. Create a new project (file) on Prism(Motul-
sky 1996). For each point on the saturation-
½F ¼ ½TA  ½SB: ð11Þ
binding curve, enter the concentration of
16 Kd and Bmax could be calculated using a Scatchard ligand into the X column and [SB] into the
plot, Woolf plot or the software. Y column.

152 | December 2015 | Volume 1 | Issue 3  The Author(s) 2016. This article is published with open access at Springerlink.com
Radioligand binding for quantitative analysis of ligand-receptor interactions PROTOCOL

ii. Select ‘‘One site binding’’ under the ‘‘Nonlin- A

ear Regression dialog’’ box to analyze the 1500
data and produce a binding curve.
1200
iii. Prism displays the best-fit values (Bmax and
Kd) for the binding parameters in the results 900
sheets. 600
300
[TIMING]
0
Step 1–3 Preparation of the 125I-Protein L and cold
ligands takes approximately 1 h. 0 50 100 150 200
Step 4–6 Preparation of the receptor samples takes B
approximately 1 h. 1500
Step 7–13 The cell-binding assay usually takes 1200
4–6 h, depending on how many samples 900
are used. 600
Step 13–16 Activity measurements and data analyses
300
take approximately 1–2 h.
0
[? TROUBLESHOOTING]
0 50 100 150 200
Step 6 The presence of certain metal ions (e.g., Mn2?
and Mg2?) in the cell-binding buffer is
essential for receptor binding. Binding buffer Fig. 1 ITLC analysis of 125I-labeled Nimotuzumab. The labeling
without these ions will result in low counts yield of 125I-Nimotuzumab was 97.6%, and the radiochemical
from the collected membrane. purity was 98.5% after purification
Step 7 It is necessary to add the cells and buffer with
multiple-channel pipettes to reduce the time of
this step. It takes practice to become skilled at shows a typical ‘‘Woolf plot’’. In parallel comparisons
adding serial concentrations of radioligand and from the same data obtained from the binding assays,
cold ligand. It is important to stay focused and the Scatchard plot, the Woolf plot and the saturation
patient. radioligand-binding curve gave similar estimates of the
Step 8 The non-specific binding assay requires a large Kd (7.81, 7.935 and 8.095 pL/mol, respectively) and
quantity of the cold ligand. Usually the ligands are Bmax (113.4, 114.5 and 115.3 pmol/L) (Table 3). The
difficult to prepare or very expensive. The average total number of EGF receptors on each UM-SCC-
Scatchard plot and the Woolf plot could be 22B cell could be calculated:
completed using fewer concentrations and fewer
115:3 pM  200 lL  6:02  1023
parallel samples. In total, about 20 samples are EGFRS per cell ¼
sufficient for fitting the linear regression. 1  105
5
¼ 1:4  10 :

ANTICIPATED RESULTS
MATERIALS
Figures 1 and 2 present typical representative data
obtained using the method described here. EGFR over- Reagents
expressing UM-SCC-22B cells were assayed against the
radiolabeled antibody 125I-Nimotuzumab. The labeling • 0.2 mol/L phosphate buffer, pH 7.4 (NaH2PO42H2O
yield of 125I-Nimotuzumab was 97.6% and the radio- 0.4063 g, Na2HPO412H2O 5.8077 g in 200 mL
chemical purity was [98.5% after purification (Fig. 1). ddH2O)
The specific activity was 24.7 Ci/g. • Chloramine-T (100 lg in 0.2 mol/L PB)
Figure 2A shows an example of a typical equilibrium • Na2S2O5 (200 lg in 100 lL ddH2O)
saturation curve using the radiolabeled assay with • KI (1 mg in 100 lL ddH2O)
increasing concentrations of 125I-Nimotuzumab. Fig- • Chloroform
ure 2B shows a typical ‘‘Scatchard plot’’. Figure 2C • Iodogen (0.5 lg/lL in chloroform)

 The Author(s) 2016. This article is published with open access at Springerlink.com 153 | December 2015 | Volume 1 | Issue 3
PROTOCOL C. Dong et al.

A 20 B 0.4

15 0.3

[SB]/[F]

[F]/[SB]
10 0.2

5 0.1

0 0.0
0 20 40 60 80 100 0 10 20 30 40
[RL] [L]
C 100

80

60
[SB]

40

20

0
0 10 20 30
[RL]

Fig. 2 The calculation of Kd and Bmax by Scatchard plot (A), Woolf plot (B) or the software (C)

Table 3 Comparison of the results obtained by three plots • Dry bath incubator (Fisher Scientific, cat. no. 11-718-
Scatchard plot Woolf plot Saturation binding curve
2)
• c-counter (PerkinElmer, 2470 automatic gamma
Kd 7.81 7.935 8.095 counter)
Bmax 113.4 114.5 115.3 • Glass-bottomed screw cap vial (Agilent Technologies,
cat. no. 5182-0715)
• Cell-binding buffer (20 mmol/L Tris, 150 mmol/L • GraphPad Prism (GraphPad Software Inc.)
NaCl, 2 mmol/L CaCl2, 1 mmol/L MnCl2, 1 mmol/L
MgCl2, 1% (wt/vol) bovine serum albumin; pH 7.4)
Reagent setup
• PBS buffer (NaH2PO42H2O 0.24 g, Na2HPO412H2O
2.901 g and NaCl 8.5 g in 1 L ddH2O)
Cell sample preparation Culture receptor-overexpressed
• Receptor-overexpressed cells (see ‘‘Reagent setup’’
cells in corresponding medium under certain condi-
section)
tions. Collect the cells from the flasks or the plates. At
• Medium
least 105 cells are needed for each well. It takes 96 wells
• Fetal bovine serum
to test the binding of one ligand with its receptor. Wash
• Na125I (Perkin Elmer, Waltham, MA, USA)
the cell solution with 0.01 mol/L sterile PBS three
times. Carefully resuspend the cells in cell binding
Equipment buffer to a concentration of 2 9 106 cells/mL.
Cold protein L preparation 500 mg of protein L is
• pH paper (Aladdin Inc.) dissolved in or diluted with 2.5 mL cell binding buffer.
• PD MidiTrap G-25 column (GE Healthcare, cat. no. However, it takes a large amount of ligand to finish the
28-9180-08) experiment. In general, the cold ligands should be 1000
• MultiScreenTM Vacuum Manifold 96-well plate (Mil- times more concentrated than the radiolabeled ligand to
lipore, cat. no. MAVM0960R) block the receptors. Fewer cold ligands could be used in
• Vacuum pump (Zhengzhou Greatwall Inc., SHB-III) low concentrations. Moreover, fewer concentrations (for

154 | December 2015 | Volume 1 | Issue 3  The Author(s) 2016. This article is published with open access at Springerlink.com
Radioligand binding for quantitative analysis of ligand-receptor interactions PROTOCOL

example, eight concentrations) could conserve many Carpenter JW, Laethem C, Hubbard FR, Eckols TK, Baez M, McClure
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between the detected cpm value and the objective cpm Chem 21:1769–1773
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Acknowledgments This work was supported by National Nat- imental biology and medicine society for experimental
ural Science Foundation of China (81125011, 81420108019 and biology and medicine, New York, vol 133, pp 989–992
81427802). Keen M (1995) The problems and pitfalls of radioligand binding.
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Klotz IM (1985) ligand-receptor interactions: facts and fantasies.
Conflict of Interest Chengyan Dong, Zhaofei Liu and Fan Wang Q Rev Biophys 18:227–259
declare that they have no conflict of interest. Maguire JJ, Kuc RE, Davenport AP (2012) Radioligand binding
assays and their analysis. In: Davis L (ed) Methods in
Human and Animal Rights and Informed Consent This article molecular biology, vol 897. Humana Press Inc, Clifton,
does not contain any studies with human or animal subjects pp 31–77
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Creative Commons Attribution 4.0 International License (http:// pp 17–55
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