introduces issues in terms of the limit of fluorescence detection
since the fluorescence of a very high-affinity probe will not be
detectable at concentrations around its K d in many instruments. Often, a suitable probe has a K d of between 1 and
10 nM. Such a probe allows reduction of the amount of target
protein required and allows for the detection of relatively highaffinity displacing compounds.
3.3.3 Measurement
of the Time to Equilibrium
Before a classic receptor/ligand equilibrium binding experiment
can be completed, the incubation period required for equilibrium
to be achieved must be empirically determined. The time taken to
reach equilibrium at room temperature, which is used for highthroughput measurements, is dependent on the concentrations of
the ligand and receptor. By using a constant label concentration and
the lowest measurable receptor concentration, it is possible to
determine the time the system takes to reach equilibrium as follows:
1. Ensure that the background from the buffer is <5% of that of
the ligand.
2. Add the ligand, at a concentration close to its K d , to the plate
and take several readings to determine the polarization of the
free ligand.
3. To the same well, add an amount of receptor approximately
equal to 1/20 of the expected K d . Since the rate of association
is dependent on the receptor concentration, this combination
of low ligand and receptor concentrations represents the longest time necessary to reach equilibrium.
4. Follow the increase in polarization over time and determine the
time at which the polarization values reach a plateau, representing the approach to equilibrium. Depending upon the binding
kinetics, the time to equilibrium may be many hours, but for
practical purposes, a suitable ligand for high-throughput
screening should reach equilibrium faster than about 5 h.
This time should then be used as the incubation period for
the binding experiments.
3.3.4 Effect of Binding
on Total Fluorescence
Once a buffer system and probe have been identified, it is important
to measure the effect of binding on the total fluorescence of the
ligand, since binding may cause a reduction in the amount of
fluorescence detected due to quenching effects:
1. Ensure that the background from the buffer is <5% of that of
the ligand.
2. Add the ligand, at a concentration close to its K d , to ten wells in
a black microtiter plate and dilute to the required assay volume
with assay buffer.
Ligand Discovery - Fluorescence Polarization
237
since the fluorescence of a very high-affinity probe will not be
detectable at concentrations around its K d in many instruments. Often, a suitable probe has a K d of between 1 and
10 nM. Such a probe allows reduction of the amount of target
protein required and allows for the detection of relatively highaffinity displacing compounds.
3.3.3 Measurement
of the Time to Equilibrium
Before a classic receptor/ligand equilibrium binding experiment
can be completed, the incubation period required for equilibrium
to be achieved must be empirically determined. The time taken to
reach equilibrium at room temperature, which is used for highthroughput measurements, is dependent on the concentrations of
the ligand and receptor. By using a constant label concentration and
the lowest measurable receptor concentration, it is possible to
determine the time the system takes to reach equilibrium as follows:
1. Ensure that the background from the buffer is <5% of that of
the ligand.
2. Add the ligand, at a concentration close to its K d , to the plate
and take several readings to determine the polarization of the
free ligand.
3. To the same well, add an amount of receptor approximately
equal to 1/20 of the expected K d . Since the rate of association
is dependent on the receptor concentration, this combination
of low ligand and receptor concentrations represents the longest time necessary to reach equilibrium.
4. Follow the increase in polarization over time and determine the
time at which the polarization values reach a plateau, representing the approach to equilibrium. Depending upon the binding
kinetics, the time to equilibrium may be many hours, but for
practical purposes, a suitable ligand for high-throughput
screening should reach equilibrium faster than about 5 h.
This time should then be used as the incubation period for
the binding experiments.
3.3.4 Effect of Binding
on Total Fluorescence
Once a buffer system and probe have been identified, it is important
to measure the effect of binding on the total fluorescence of the
ligand, since binding may cause a reduction in the amount of
fluorescence detected due to quenching effects:
1. Ensure that the background from the buffer is <5% of that of
the ligand.
2. Add the ligand, at a concentration close to its K d , to ten wells in
a black microtiter plate and dilute to the required assay volume
with assay buffer.
Ligand Discovery - Fluorescence Polarization
237