2.1.8 Filter-Binding Assay/Filter Retention Assay [4, 13]
Filter-binding assays quantify the signal of a fluorescently or radioactively labeled
aptamer binding to a target that is immobilized on a filter membrane. Reading out
different concentration steps of fluorescent aptamers allows for determining the
equilibrium-binding affinity (K D ).
In a typical filter-binding assay, one interaction partner (usually the aptamer
target) is immobilized on a membrane, whereas the second interaction partner
(usually the aptamer) is titrated across a certain concentration range.
2.1.9 Electromobility Shift Assay (EMSA) [14, 15]
EMSA monitors mobility differences between complexed and unbound molecules in
net-like matrices or gels, in order to determine the equilibrium-binding affinity (K D )
between the interaction partners.
In a typical EMSA experiment, a constant concentration of a labeled aptamer is
incubated with increasing concentrations of its target. The mixed samples are loaded
on a gel matrix in order to separate complexes from unbound aptamers by size. A
fluorescent or radioactive readout allows for quantifying the interaction strength.
2.2 Sample Material Requirements of the Selected
Biophysical Methods
Besides the fact that the data quality of any biophysical method is enhanced with
increased purity, homogeneity, stability, solubility, and reduced aggregation tendency of the sample material, every method has its specific prerequisites and
requirements to the sample material due to its specific principle and technical setup.
Surface-based methods, such as SPR, BLI, and SwitchSENSE, require immobilization of one interaction partner to the sensor surface. In SPR and BLI, various
immobilization strategies are available using direct immobilization via capturing of
epitopes already available on the target (e.g., via a hexahistidine tag on a protein).
Alternatively, chemical processes are available for linking the target either directly
(via amino acid side chains) on the sensor surface or to adaptors (such as biotin),
which are then captured on a pre-coated surface. In SwitchSENSE, a target molecule
needs to be modified with a DNA strand, which is then hybridized to a counterpart
on the sensor surface. Aptamers may directly be hybridized via extended sequences
that bind to the DNA nano-lever on the sensor surface.
In MST, flow cytometry, EMSA, FP, and filter-binding assays, one interaction
partner must be fluorescent. MST can either work with intrinsic molecule fluorescence (tryptophan fluorescence in proteins or peptides, label-free MST) or relies on
labeling one of the interaction partners with a fluorophore. In flow cytometry, the
8
M. Plach and T. Schubert
Filter-binding assays quantify the signal of a fluorescently or radioactively labeled
aptamer binding to a target that is immobilized on a filter membrane. Reading out
different concentration steps of fluorescent aptamers allows for determining the
equilibrium-binding affinity (K D ).
In a typical filter-binding assay, one interaction partner (usually the aptamer
target) is immobilized on a membrane, whereas the second interaction partner
(usually the aptamer) is titrated across a certain concentration range.
2.1.9 Electromobility Shift Assay (EMSA) [14, 15]
EMSA monitors mobility differences between complexed and unbound molecules in
net-like matrices or gels, in order to determine the equilibrium-binding affinity (K D )
between the interaction partners.
In a typical EMSA experiment, a constant concentration of a labeled aptamer is
incubated with increasing concentrations of its target. The mixed samples are loaded
on a gel matrix in order to separate complexes from unbound aptamers by size. A
fluorescent or radioactive readout allows for quantifying the interaction strength.
2.2 Sample Material Requirements of the Selected
Biophysical Methods
Besides the fact that the data quality of any biophysical method is enhanced with
increased purity, homogeneity, stability, solubility, and reduced aggregation tendency of the sample material, every method has its specific prerequisites and
requirements to the sample material due to its specific principle and technical setup.
Surface-based methods, such as SPR, BLI, and SwitchSENSE, require immobilization of one interaction partner to the sensor surface. In SPR and BLI, various
immobilization strategies are available using direct immobilization via capturing of
epitopes already available on the target (e.g., via a hexahistidine tag on a protein).
Alternatively, chemical processes are available for linking the target either directly
(via amino acid side chains) on the sensor surface or to adaptors (such as biotin),
which are then captured on a pre-coated surface. In SwitchSENSE, a target molecule
needs to be modified with a DNA strand, which is then hybridized to a counterpart
on the sensor surface. Aptamers may directly be hybridized via extended sequences
that bind to the DNA nano-lever on the sensor surface.
In MST, flow cytometry, EMSA, FP, and filter-binding assays, one interaction
partner must be fluorescent. MST can either work with intrinsic molecule fluorescence (tryptophan fluorescence in proteins or peptides, label-free MST) or relies on
labeling one of the interaction partners with a fluorophore. In flow cytometry, the
8
M. Plach and T. Schubert