the compound appears to have displaced the probe, even though it
may not have done so. In a high-throughput fluorescence polarization screen, Turconi et al. [19] found that the most common cause
of compound interference was autofluorescence. Autofluorescent
compounds may easily be identified by measuring total fluorescence
intensity. Wells where the total intensity is statistically significantly
greater than that of control wells should be flagged as potential
artifacts. It may be possible to correct for this effect where the level
of compound fluorescence is relatively low, but a preferred
approach is to employ a probe carrying a label that is fluorescent
at a wavelength that is significantly different from the wavelengths
where compounds in the collection may interfere.
Compounds may also interfere if they aggregate or precipitate
during the assay. Such aggregates or precipitates are often highly
anisotropic due to light scattering. This results in very high polarization values that could mask a true displacement effect, leading to
false-negative results.
Another potential issue arises from inner filter effects if compounds absorb the excitation or emitted light (see Note 26). However, as fluorescence polarization is a ratiometic technique, this
effect is often well tolerated, and checks can be made by carrying
out absorbance scans on compounds where this effect may be
suspected.
4 Notes
1. Many biochemical interactions are affected by changes in
pH. The H
+ concentration in vitro must therefore be controlled by adding a suitable buffer to the medium, without
affecting the function of the system. A buffer keeps the pH of
a solution constant by taking up protons when released during
reactions, or by releasing protons when they are consumed by
reactions.
2. The buffer capacity represents the amount of H
+ or OH
À ions
that can be neutralized by the buffer. The buffer capacity is
related to the buffer concentration and the pH at which it is
used relative to the buffer pK a value. Generally, buffers may
only be reliably used within a pH range of one pH unit above or
below the pK a .
3. HEPES is a member of the list of 12 buffers produced by
Norman Good [20] and has favorable qualities for biochemical
assay development. HEPES has a pK a of 7.55, at 20
C, which
is close to physiological pH. Tris is also often used but has poor
buffering capacity below pH 7.5. Care is needed to ensure that
the pH is measured at the temperature at which the buffer will
be used, as pK a varies with temperature.
242
Geoffrey A. Holdgate and Paul E. Hemsley
may not have done so. In a high-throughput fluorescence polarization screen, Turconi et al. [19] found that the most common cause
of compound interference was autofluorescence. Autofluorescent
compounds may easily be identified by measuring total fluorescence
intensity. Wells where the total intensity is statistically significantly
greater than that of control wells should be flagged as potential
artifacts. It may be possible to correct for this effect where the level
of compound fluorescence is relatively low, but a preferred
approach is to employ a probe carrying a label that is fluorescent
at a wavelength that is significantly different from the wavelengths
where compounds in the collection may interfere.
Compounds may also interfere if they aggregate or precipitate
during the assay. Such aggregates or precipitates are often highly
anisotropic due to light scattering. This results in very high polarization values that could mask a true displacement effect, leading to
false-negative results.
Another potential issue arises from inner filter effects if compounds absorb the excitation or emitted light (see Note 26). However, as fluorescence polarization is a ratiometic technique, this
effect is often well tolerated, and checks can be made by carrying
out absorbance scans on compounds where this effect may be
suspected.
4 Notes
1. Many biochemical interactions are affected by changes in
pH. The H
+ concentration in vitro must therefore be controlled by adding a suitable buffer to the medium, without
affecting the function of the system. A buffer keeps the pH of
a solution constant by taking up protons when released during
reactions, or by releasing protons when they are consumed by
reactions.
2. The buffer capacity represents the amount of H
+ or OH
À ions
that can be neutralized by the buffer. The buffer capacity is
related to the buffer concentration and the pH at which it is
used relative to the buffer pK a value. Generally, buffers may
only be reliably used within a pH range of one pH unit above or
below the pK a .
3. HEPES is a member of the list of 12 buffers produced by
Norman Good [20] and has favorable qualities for biochemical
assay development. HEPES has a pK a of 7.55, at 20
C, which
is close to physiological pH. Tris is also often used but has poor
buffering capacity below pH 7.5. Care is needed to ensure that
the pH is measured at the temperature at which the buffer will
be used, as pK a varies with temperature.
242
Geoffrey A. Holdgate and Paul E. Hemsley