19. For dilution of the target protein, it is very useful if the stock
concentration is at a relatively high concentration so that additional components in the storage buffer may be diluted out.
20. As a guide, if the probe can be detected at a concentration of
0.3 nM, it is only necessary to dilute the target to around
0.2 nM.
21. Carrying out intermediate dilutions in DMSO avoids decreasing the solvent concentration by the addition of buffer until
the final dilution, which helps to maintain compound solubility. The final DMSO concentration is dependent on the effect
of DMSO on the assay, but it is useful to keep it at or below 1%
(v/v). It is also important to remember that the addition of
competitor compounds will introduce a further amount of
DMSO.
22. At this stage, it is also prudent to test the tolerance of the assay
to different concentrations of DMSO, linked to Note 21
above.
23. Often, a compound known to fully displace the probe is used
for the measurement of this Min value.
24. The Hill slope refers to the steepness of the curve and often
reflects the stoichiometry of the interaction.
25. IC 50 -to-K i : A web-based tool for converting IC 50 to K i values
for inhibitors of enzyme activity and ligand binding [22]—
https://bioinfo-abcc.ncifcrf.gov/IC50_Ki_Converter/index.
php.
26. Inner filter effects can be classed as a primary inner filter
effect—defined as the decrease in the intensity of the excitation
light because of optical absorption in the excitation region, or
secondary inner filter effect when the fluorescence intensity
decreases because of the absorption in the emission region.
For dilute solutions (where absorbance <0.01), the effect is
negligible.
References
1. Powell D, Hertzberg RP, Macarro ´ n R (2016)
Design and implementation of highthroughput screening assays. Methods Mol
Biol 1439:1–32
2. Janzen W (2014) Screening technologies for
small molecule discovery: the state of the art.
Chem Biol 21:1162–1170
3. Macarro ´ n R, Banks MN, Bojanic D et al (2011)
Impact of high-throughput screening in biomedical research. Nat Rev Drug Discov
10:188–195
4. Mayr L, Bojanic D (2009) Novel trends in
high-throughput screening. Curr Opin Pharmacol 9:580–588
5. Burke TJ, Loniello KR, Beebe JA, Ervin KM
(2003) Development and application of fluorescence polarization assays in drug discovery.
Comb Chem High Throughput Screen
6:183–194
6. Owicki J (2000) Fluorescence polarization and
anisotropy in high throughput screening: perspectives and primer. J Biomol Screen
5:297–306
Ligand Discovery - Fluorescence Polarization
245
concentration is at a relatively high concentration so that additional components in the storage buffer may be diluted out.
20. As a guide, if the probe can be detected at a concentration of
0.3 nM, it is only necessary to dilute the target to around
0.2 nM.
21. Carrying out intermediate dilutions in DMSO avoids decreasing the solvent concentration by the addition of buffer until
the final dilution, which helps to maintain compound solubility. The final DMSO concentration is dependent on the effect
of DMSO on the assay, but it is useful to keep it at or below 1%
(v/v). It is also important to remember that the addition of
competitor compounds will introduce a further amount of
DMSO.
22. At this stage, it is also prudent to test the tolerance of the assay
to different concentrations of DMSO, linked to Note 21
above.
23. Often, a compound known to fully displace the probe is used
for the measurement of this Min value.
24. The Hill slope refers to the steepness of the curve and often
reflects the stoichiometry of the interaction.
25. IC 50 -to-K i : A web-based tool for converting IC 50 to K i values
for inhibitors of enzyme activity and ligand binding [22]—
https://bioinfo-abcc.ncifcrf.gov/IC50_Ki_Converter/index.
php.
26. Inner filter effects can be classed as a primary inner filter
effect—defined as the decrease in the intensity of the excitation
light because of optical absorption in the excitation region, or
secondary inner filter effect when the fluorescence intensity
decreases because of the absorption in the emission region.
For dilute solutions (where absorbance <0.01), the effect is
negligible.
References
1. Powell D, Hertzberg RP, Macarro ´ n R (2016)
Design and implementation of highthroughput screening assays. Methods Mol
Biol 1439:1–32
2. Janzen W (2014) Screening technologies for
small molecule discovery: the state of the art.
Chem Biol 21:1162–1170
3. Macarro ´ n R, Banks MN, Bojanic D et al (2011)
Impact of high-throughput screening in biomedical research. Nat Rev Drug Discov
10:188–195
4. Mayr L, Bojanic D (2009) Novel trends in
high-throughput screening. Curr Opin Pharmacol 9:580–588
5. Burke TJ, Loniello KR, Beebe JA, Ervin KM
(2003) Development and application of fluorescence polarization assays in drug discovery.
Comb Chem High Throughput Screen
6:183–194
6. Owicki J (2000) Fluorescence polarization and
anisotropy in high throughput screening: perspectives and primer. J Biomol Screen
5:297–306
Ligand Discovery - Fluorescence Polarization
245