that background fluorescence is not significant, rather than to
correct for it. Two approaches to keep background fluorescence
as low as possible are to use black microtiter plates (see Note 14)
and to use red-shifted fluorophores (see Note 15).
Background fluorescence that is less than 5% of the ligand at its
final assay concentration can be ignored. If background fluorescence cannot be removed due to the requirement for a critical assay
component, then the best approach is to collect individual fluorescence values from each detector/plane, subtract the background
fluorescence value, and use these corrected values in the polarization calculation (see Subheading 3.3.4).
Another reason for measuring background fluorescence in the
buffer is to determine whether it is equal in each detector/plane. A
significant difference in the two values is indicative of the presence
of a polarizing component in the buffer. Often, this may be indicative of particulates that are known to produce highly polarized
scattered light, the presence of which may be remedied by filtering
the buffer.
3.3.2 Selection of a
Binding Probe
Fluorescent probe selection is, arguably, the most difficult step in
the development of a good FP assay. Protein targets that are well
known in the literature will often have known small molecule
binders, which can be chemically modified to produce a fluorescent
binding probe. A good probe will have all of the properties
described below:
1. A red-shifted absorbance wavelength. It is crucial that compounds do not interfere in the assay either by fluorescing or
quenching at the wavelengths used. This is because FP is a
technology that lends itself very well to measuring the effects
of compounds in a homogeneous mixture without separation
steps. For high-throughput screening, the Alexa 647
® dye is
often used as the wavelengths used are longer than most of the
compounds in the screening collection. A way of measuring
and dealing with fluorescent interference is described in Subheading 3.3.4.
2. High fluorescence yield. The probe should have a high fluorescence quantum yield so that it may be detectable in low
concentrations.
3. Suitable binding affinity. A good probe should bind with an
affinity that is neither too high nor too low. Low-affinity probes
require a relatively high concentration of target protein to
achieve the level of binding required to get a high polarization
signal. This is problematic in terms of protein reagent consumption and results in sensitivity issues due to the lower
limit on the measurable IC 50 that is introduced by the target
protein concentration (see Note 16). A high-affinity probe
236
Geoffrey A. Holdgate and Paul E. Hemsley
correct for it. Two approaches to keep background fluorescence
as low as possible are to use black microtiter plates (see Note 14)
and to use red-shifted fluorophores (see Note 15).
Background fluorescence that is less than 5% of the ligand at its
final assay concentration can be ignored. If background fluorescence cannot be removed due to the requirement for a critical assay
component, then the best approach is to collect individual fluorescence values from each detector/plane, subtract the background
fluorescence value, and use these corrected values in the polarization calculation (see Subheading 3.3.4).
Another reason for measuring background fluorescence in the
buffer is to determine whether it is equal in each detector/plane. A
significant difference in the two values is indicative of the presence
of a polarizing component in the buffer. Often, this may be indicative of particulates that are known to produce highly polarized
scattered light, the presence of which may be remedied by filtering
the buffer.
3.3.2 Selection of a
Binding Probe
Fluorescent probe selection is, arguably, the most difficult step in
the development of a good FP assay. Protein targets that are well
known in the literature will often have known small molecule
binders, which can be chemically modified to produce a fluorescent
binding probe. A good probe will have all of the properties
described below:
1. A red-shifted absorbance wavelength. It is crucial that compounds do not interfere in the assay either by fluorescing or
quenching at the wavelengths used. This is because FP is a
technology that lends itself very well to measuring the effects
of compounds in a homogeneous mixture without separation
steps. For high-throughput screening, the Alexa 647
® dye is
often used as the wavelengths used are longer than most of the
compounds in the screening collection. A way of measuring
and dealing with fluorescent interference is described in Subheading 3.3.4.
2. High fluorescence yield. The probe should have a high fluorescence quantum yield so that it may be detectable in low
concentrations.
3. Suitable binding affinity. A good probe should bind with an
affinity that is neither too high nor too low. Low-affinity probes
require a relatively high concentration of target protein to
achieve the level of binding required to get a high polarization
signal. This is problematic in terms of protein reagent consumption and results in sensitivity issues due to the lower
limit on the measurable IC 50 that is introduced by the target
protein concentration (see Note 16). A high-affinity probe
236
Geoffrey A. Holdgate and Paul E. Hemsley