3.3 Experimental
Design and Assay
Development
FP measurements report the status of a binding interaction and may
be used in several different ways to provide information about the
behavior of small molecule drugs. One of the primary uses is to
identify compounds that can displace a fluorescent probe from a
target protein, and it this type of approach that will be described
below. However, many of the experimental approaches toward
assay development in this format will be equally appropriate for
the development of other formats.
3.3.1 Measurement
of Background
Fluorescence
Instruments report fluorescence intensity in both planes as well as
the calculated ratiometric FP value (see Note 13). It is important to
measure the fluorescence properties of the buffer that has been
optimized because significant background fluorescence should be
subtracted from each detector/plane prior to the calculation of the
polarization value. Most instruments do not do this, and they
simply return values of individual fluorescence values and the calculated polarization value. For this reason, it is often easier to ensure
Polarisation film
horizontal
Excitation light
Excitation filter
Emission light
Emission light
Polarisation film
vertical
Polarisation film
horizontal
Emission filter
Emission filter
Dichroic mirror
Dual emission beam splitter
Fig. 1 Schematic representation of a dual detector instrument
Ligand Discovery - Fluorescence Polarization
235
Design and Assay
Development
FP measurements report the status of a binding interaction and may
be used in several different ways to provide information about the
behavior of small molecule drugs. One of the primary uses is to
identify compounds that can displace a fluorescent probe from a
target protein, and it this type of approach that will be described
below. However, many of the experimental approaches toward
assay development in this format will be equally appropriate for
the development of other formats.
3.3.1 Measurement
of Background
Fluorescence
Instruments report fluorescence intensity in both planes as well as
the calculated ratiometric FP value (see Note 13). It is important to
measure the fluorescence properties of the buffer that has been
optimized because significant background fluorescence should be
subtracted from each detector/plane prior to the calculation of the
polarization value. Most instruments do not do this, and they
simply return values of individual fluorescence values and the calculated polarization value. For this reason, it is often easier to ensure
Polarisation film
horizontal
Excitation light
Excitation filter
Emission light
Emission light
Polarisation film
vertical
Polarisation film
horizontal
Emission filter
Emission filter
Dichroic mirror
Dual emission beam splitter
Fig. 1 Schematic representation of a dual detector instrument
Ligand Discovery - Fluorescence Polarization
235