on the inverse relationship between molecular rotation and the
degree of polarization of a fluorophore excited by linear polarized
light [7]. As the apparent molecular weight of the fluorescent probe
molecule is increased by binding to the target protein, the polarization of the subsequent emitted light increases. This phenomenon
has been exploited to configure assays for several different
biological targets in a single molecule binding (protein–ligand
interaction) assays through enzyme activity (substrate turnover to
product) and even to live-cell formats [8, 9]. There are several
advantages of FP assays that make them particularly applicable to
high-throughput screening, including solution-based measurement, the ability to measure kinetics, the avoidance of radioisotopes, the simplicity of the assay format, the lack of filtration or
separation steps, and the relatively inexpensive reagents required.
Additionally, as FP is a ratiometric measurement (it is independent
of the actual light intensity and hence the fluorophore concentration because it is defined as a ratio of two components, both of
which are themselves proportional to the concentration), it is relatively insensitive to artifacts such as compound absorption or inner
filter effects that may constrain other light-based technologies.
However, the technique can suffer from some issues experienced
by other methods such as light scattering and autofluorescence
[10]. The technology is readily scalable to both 384- and 1536well formats, and with improvements in the understanding of issues
associated with fluorescent probes (including peptides, small molecule drugs, and cytokines) used in FP assays, such as the potential
for depolarization due to flexibility in the attachment of the dye,
sometimes referred to as the “propeller effect,” suitable probes
utilizing dyes such as fluorescein, rhodamine, BODIPY, Cy, and
Alexa fluors, without long aliphatic linkers, can often be synthesized [11]. In this chapter, we will predominantly use the terminology for fluorescence polarization, using anisotropy in equations,
where this is simpler.
2 Materials
Buffer systems (see Note 1) will vary depending upon the binding
interaction studied. Ideally, buffer systems should be as simple as
possible, containing the minimum number of additives required to
support the functional biochemical interaction. Buffers should be
chosen such that a suitable buffering capacity is available to minimize changes in pH during the reaction of interest (see Note 2).
Often, buffers such as Tris or HEPES (see Note 3) and additives
such as inorganic salts containing magnesium or sodium ions (see
Note 4), as well as reducing agents including DTT and TCEP (see
Note 5), are suitable for FP assays. Subcritical micellar concentrations (CMCs) of detergents are also frequently used (see Note 6).
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Geoffrey A. Holdgate and Paul E. Hemsley
degree of polarization of a fluorophore excited by linear polarized
light [7]. As the apparent molecular weight of the fluorescent probe
molecule is increased by binding to the target protein, the polarization of the subsequent emitted light increases. This phenomenon
has been exploited to configure assays for several different
biological targets in a single molecule binding (protein–ligand
interaction) assays through enzyme activity (substrate turnover to
product) and even to live-cell formats [8, 9]. There are several
advantages of FP assays that make them particularly applicable to
high-throughput screening, including solution-based measurement, the ability to measure kinetics, the avoidance of radioisotopes, the simplicity of the assay format, the lack of filtration or
separation steps, and the relatively inexpensive reagents required.
Additionally, as FP is a ratiometric measurement (it is independent
of the actual light intensity and hence the fluorophore concentration because it is defined as a ratio of two components, both of
which are themselves proportional to the concentration), it is relatively insensitive to artifacts such as compound absorption or inner
filter effects that may constrain other light-based technologies.
However, the technique can suffer from some issues experienced
by other methods such as light scattering and autofluorescence
[10]. The technology is readily scalable to both 384- and 1536well formats, and with improvements in the understanding of issues
associated with fluorescent probes (including peptides, small molecule drugs, and cytokines) used in FP assays, such as the potential
for depolarization due to flexibility in the attachment of the dye,
sometimes referred to as the “propeller effect,” suitable probes
utilizing dyes such as fluorescein, rhodamine, BODIPY, Cy, and
Alexa fluors, without long aliphatic linkers, can often be synthesized [11]. In this chapter, we will predominantly use the terminology for fluorescence polarization, using anisotropy in equations,
where this is simpler.
2 Materials
Buffer systems (see Note 1) will vary depending upon the binding
interaction studied. Ideally, buffer systems should be as simple as
possible, containing the minimum number of additives required to
support the functional biochemical interaction. Buffers should be
chosen such that a suitable buffering capacity is available to minimize changes in pH during the reaction of interest (see Note 2).
Often, buffers such as Tris or HEPES (see Note 3) and additives
such as inorganic salts containing magnesium or sodium ions (see
Note 4), as well as reducing agents including DTT and TCEP (see
Note 5), are suitable for FP assays. Subcritical micellar concentrations (CMCs) of detergents are also frequently used (see Note 6).
232
Geoffrey A. Holdgate and Paul E. Hemsley