4. Metal ions are chosen where they are needed as cofactors and
are added at concentrations to mimic the physiological environment of the target. Experiments conducted during assay
development determine the optimum concentration of
cofactors.
5. Reducing agents are used to reproduce the reducing environment within cells and to prevent disulfide bond formation and
potential aggregation of cytosolic proteins.
6. Detergents are added to help prevent protein adherence to
plastic as well as to solubilize test compounds and to prevent
their aggregation. Nonionic detergents (Tween 20, Triton
X-100, BRIJ, Octyl β-D-glucopyranoside, etc.) are often preferred, as they are less harsh and are nondenaturing compared
with ionic detergents. Thus, they are often employed when
maintaining protein structure is important. The choice of the
optimal detergent and the concentration for use is made during
assay development.
7. Buffer components may be prepared by sterile filtration
(through a 0.22 μm filter) or by autoclaving. Heat-labile components should not be autoclaved. The literature has mixed
opinions on whether HEPES can be safely autoclaved.
8. The high-molecular-weight binding partner should be physiologically relevant and as pure as possible to minimize the presence of nonspecific binding sites. Knowledge of the number
and nature of binding sites on each molecule is useful, and
analysis is simplified when there is a single site.
9. The fluorescent low-molecular-weight binding partner can
often be identified from the literature. Alternatively, chemical
fluorescence labeling of known tool compounds can be undertaken. Reference 8 contains several examples of the generation
of FP probes using several different fluorescent dyes. Fluorescence labeling of tool compounds will frequently yield a molecule that will still bind to the target protein but with different
affinity compared to the parent molecule. For high-throughput
screening applications, such tool compounds are preferentially
labeled with dyes such as Alexa647
® because the excitation and
emission wavelengths are in the far-red part of the electromagnetic spectrum, where interference due to compound absorption is significantly reduced. The Alexa Fluor family of
fluorescent dyes is a series of dyes invented by Molecular Probes
(now part of Thermo Fisher Scientific and sold under the
Invitrogen brand name). The dyes can be conjugated directly
to a range of biomolecules.
10. Buffer is drawn into a syringe, an acrodisc (or similar 0.22-μm
filter) is added, and the syringe contents are filtered into a
sterile container.
Ligand Discovery - Fluorescence Polarization
243
are added at concentrations to mimic the physiological environment of the target. Experiments conducted during assay
development determine the optimum concentration of
cofactors.
5. Reducing agents are used to reproduce the reducing environment within cells and to prevent disulfide bond formation and
potential aggregation of cytosolic proteins.
6. Detergents are added to help prevent protein adherence to
plastic as well as to solubilize test compounds and to prevent
their aggregation. Nonionic detergents (Tween 20, Triton
X-100, BRIJ, Octyl β-D-glucopyranoside, etc.) are often preferred, as they are less harsh and are nondenaturing compared
with ionic detergents. Thus, they are often employed when
maintaining protein structure is important. The choice of the
optimal detergent and the concentration for use is made during
assay development.
7. Buffer components may be prepared by sterile filtration
(through a 0.22 μm filter) or by autoclaving. Heat-labile components should not be autoclaved. The literature has mixed
opinions on whether HEPES can be safely autoclaved.
8. The high-molecular-weight binding partner should be physiologically relevant and as pure as possible to minimize the presence of nonspecific binding sites. Knowledge of the number
and nature of binding sites on each molecule is useful, and
analysis is simplified when there is a single site.
9. The fluorescent low-molecular-weight binding partner can
often be identified from the literature. Alternatively, chemical
fluorescence labeling of known tool compounds can be undertaken. Reference 8 contains several examples of the generation
of FP probes using several different fluorescent dyes. Fluorescence labeling of tool compounds will frequently yield a molecule that will still bind to the target protein but with different
affinity compared to the parent molecule. For high-throughput
screening applications, such tool compounds are preferentially
labeled with dyes such as Alexa647
® because the excitation and
emission wavelengths are in the far-red part of the electromagnetic spectrum, where interference due to compound absorption is significantly reduced. The Alexa Fluor family of
fluorescent dyes is a series of dyes invented by Molecular Probes
(now part of Thermo Fisher Scientific and sold under the
Invitrogen brand name). The dyes can be conjugated directly
to a range of biomolecules.
10. Buffer is drawn into a syringe, an acrodisc (or similar 0.22-μm
filter) is added, and the syringe contents are filtered into a
sterile container.
Ligand Discovery - Fluorescence Polarization
243