11. The polarization and anisotropy values contain the same information. The main difference is that the value I k + 2.I ⊥ is equal
to the total fluorescence of the fluorophore and accounts for
emitted light that is perpendicularly polarized in the third
dimension and is therefore not captured by any detector.
Anisotropy may often be preferred because it is normalized
by the total intensity and so simplifies some of the equations.
P has physically possible values ranging from À0.33 to 0.5,
although measured values in biochemical studies typically
range from 0.01 to 0.3 or 10 to 300 mP (mP ¼ P Â 1000).
Modern instrumentation allows very precise measurements
(P Æ 0.002 or Æ2 mP), which leads to a relatively wide dynamic
range.
It is possible to convert between anisotropy and polarization as follows:
P ¼ 3r= 2 þ r
ð
Þand r ¼ 2P= 3 À P
ð
Þ
ð3Þ
12. Instruments such as the Pherastar will, at the same time, optimize read height and fluorescence gain for each detector to
return the target mP value.
13. Ratiometric methods are based on the use of a ratio between
two fluorescence intensities. This approach allows correction of
potential interference due to artifacts, bleaching, and issues
with variation in changes in focus or laser intensity.
14. Black plates help to avoid strong fluorescence signals affecting
adjacent wells, sometimes referred to as a halo effect.
15. The fluorescence profile of more than 70,000 samples across
spectral regions commonly utilized in HTS was analyzed by
Simeonov et al. [21]. It was demonstrated that red-shifting the
spectral window by as little as 100 nm was accompanied by a
dramatic decrease in autofluorescence.
16. There is a practical limit to the IC 50 that can be measured in
any FP assay. This limit is equal to [target protein]/2.
17. In practice, the effect of binding driven quenching is an exponential function (Fig. 2).
When measuring compound antagonism, the corrected
anisotropy shifts make no difference to the measured IC 50
value. However, the correct binding K d for the ligand binding
to the receptor must be known to correctly convert a measured
IC 50 value to the desired K i value.
18. Competition experiments between the probe and test compounds reveal compounds which can displace the probe, and
concentration responses allow the measurement of an IC 50
value. To calculate the true affinity of the test compound
requires knowledge of the probe K d value and application of
a relevant equation (see Note 25).
244
Geoffrey A. Holdgate and Paul E. Hemsley
to the total fluorescence of the fluorophore and accounts for
emitted light that is perpendicularly polarized in the third
dimension and is therefore not captured by any detector.
Anisotropy may often be preferred because it is normalized
by the total intensity and so simplifies some of the equations.
P has physically possible values ranging from À0.33 to 0.5,
although measured values in biochemical studies typically
range from 0.01 to 0.3 or 10 to 300 mP (mP ¼ P Â 1000).
Modern instrumentation allows very precise measurements
(P Æ 0.002 or Æ2 mP), which leads to a relatively wide dynamic
range.
It is possible to convert between anisotropy and polarization as follows:
P ¼ 3r= 2 þ r
ð
Þand r ¼ 2P= 3 À P
ð
Þ
ð3Þ
12. Instruments such as the Pherastar will, at the same time, optimize read height and fluorescence gain for each detector to
return the target mP value.
13. Ratiometric methods are based on the use of a ratio between
two fluorescence intensities. This approach allows correction of
potential interference due to artifacts, bleaching, and issues
with variation in changes in focus or laser intensity.
14. Black plates help to avoid strong fluorescence signals affecting
adjacent wells, sometimes referred to as a halo effect.
15. The fluorescence profile of more than 70,000 samples across
spectral regions commonly utilized in HTS was analyzed by
Simeonov et al. [21]. It was demonstrated that red-shifting the
spectral window by as little as 100 nm was accompanied by a
dramatic decrease in autofluorescence.
16. There is a practical limit to the IC 50 that can be measured in
any FP assay. This limit is equal to [target protein]/2.
17. In practice, the effect of binding driven quenching is an exponential function (Fig. 2).
When measuring compound antagonism, the corrected
anisotropy shifts make no difference to the measured IC 50
value. However, the correct binding K d for the ligand binding
to the receptor must be known to correctly convert a measured
IC 50 value to the desired K i value.
18. Competition experiments between the probe and test compounds reveal compounds which can displace the probe, and
concentration responses allow the measurement of an IC 50
value. To calculate the true affinity of the test compound
requires knowledge of the probe K d value and application of
a relevant equation (see Note 25).
244
Geoffrey A. Holdgate and Paul E. Hemsley