It should be noted that this correction is only needed for
severely quenched ligands, and, in these circumstances, the correction is very sensitive in that small changes in measured anisotropy
can lead to large changes in corrected anisotropy and for this reason
measured anisotropy values must be measured extremely carefully
(see Note 17).
If possible, it is preferable to work with ligands that are
quenched by less than 75% on binding. If the quenching is more
severe, then a correction may be applied, but the variability of the
correction at low anisotropy values means that the measurements
must be made extremely carefully. Hence, it is advisable to employ a
relatively unquenched ligand rather than to treat high levels of
quench mathematically.
3.3.5 Measurement
of Binding of the Probe
to the Target
To design experiments that demonstrate whether test compounds
can displace the probe, it is important to have a situation, in the
absence of test compound, where the probe is almost fully bound to
the target. This is so that displacement by test compound will result
in a change of anisotropy that may be measured. To arrange for this
situation and to allow subsequent calculation of test compound K i
values, the affinity of the probe must be measured (see Note 18).
To do this, a simple titration of target protein is made in the
presence of several different concentrations of the probe (see Note
19).
The target protein is diluted to $800 nM in assay buffer
followed by a serial dilution in assay buffer to give a range of target
protein concentrations. A twofold serial dilution is ideal in this
respect so that there are a large number of points on the
concentration-response curve (see Note 20). A control representing zero target protein should also be made using assay buffer
alone.
The fluorescent probe is diluted depending upon the storage
conditions of the probe. Probe concentrations of 60, 20, 6, 2, and
0.6 nM are good starting points. If the probe is stored in DMSO,
then any intermediate dilution steps required should be carried out
in DMSO before subsequent dilution to 2Â the final assay concentration using assay buffer (see Note 21). Equal volumes of target
protein and probe are mixed such that there is a concentrationresponse curve for target protein at each probe concentration. The
observed K d values will change with probe concentration where the
probe concentration is higher than its K d . As the probe is diluted to
a value below the K d concentration, the measured K d will approach
a constant value, representing the true K d (Fig. 3).
This experimental format can also be used to measure the
kinetics of probe binding. This involves reading the plate at regular
intervals over time. This provides an understanding of how long it
takes to reach equilibrium (see Subheading 3.3.3), which is
Ligand Discovery - Fluorescence Polarization
239
severely quenched ligands, and, in these circumstances, the correction is very sensitive in that small changes in measured anisotropy
can lead to large changes in corrected anisotropy and for this reason
measured anisotropy values must be measured extremely carefully
(see Note 17).
If possible, it is preferable to work with ligands that are
quenched by less than 75% on binding. If the quenching is more
severe, then a correction may be applied, but the variability of the
correction at low anisotropy values means that the measurements
must be made extremely carefully. Hence, it is advisable to employ a
relatively unquenched ligand rather than to treat high levels of
quench mathematically.
3.3.5 Measurement
of Binding of the Probe
to the Target
To design experiments that demonstrate whether test compounds
can displace the probe, it is important to have a situation, in the
absence of test compound, where the probe is almost fully bound to
the target. This is so that displacement by test compound will result
in a change of anisotropy that may be measured. To arrange for this
situation and to allow subsequent calculation of test compound K i
values, the affinity of the probe must be measured (see Note 18).
To do this, a simple titration of target protein is made in the
presence of several different concentrations of the probe (see Note
19).
The target protein is diluted to $800 nM in assay buffer
followed by a serial dilution in assay buffer to give a range of target
protein concentrations. A twofold serial dilution is ideal in this
respect so that there are a large number of points on the
concentration-response curve (see Note 20). A control representing zero target protein should also be made using assay buffer
alone.
The fluorescent probe is diluted depending upon the storage
conditions of the probe. Probe concentrations of 60, 20, 6, 2, and
0.6 nM are good starting points. If the probe is stored in DMSO,
then any intermediate dilution steps required should be carried out
in DMSO before subsequent dilution to 2Â the final assay concentration using assay buffer (see Note 21). Equal volumes of target
protein and probe are mixed such that there is a concentrationresponse curve for target protein at each probe concentration. The
observed K d values will change with probe concentration where the
probe concentration is higher than its K d . As the probe is diluted to
a value below the K d concentration, the measured K d will approach
a constant value, representing the true K d (Fig. 3).
This experimental format can also be used to measure the
kinetics of probe binding. This involves reading the plate at regular
intervals over time. This provides an understanding of how long it
takes to reach equilibrium (see Subheading 3.3.3), which is
Ligand Discovery - Fluorescence Polarization
239