level of 2‰ at “medium” MST power, the signal change upon
ligand binding should be at least 6‰ (e.g., a change from an
F norm value of 857‰ to 863‰). Both the noise and the amplitude
are displayed when reviewing the dose–response panel. In order to
measure the K d , we incubate our (fluorescently labeled) receptor, at
a concentration below the K d , with increasing concentrations of
non-labeled ligand. An easy way to do this is to perform a dilution
series, in the form of 1:1 dilutions. In the Monolith instruments,
we can use 16 concentrations; this will cover a concentration range
of 3 Â 10
4 (2
15
).
3.2.1 Sample
Preparation
Non-labeled Ku and DNA are prepared as described in Subheading
3.1.2. First, always spin down the stocks for 5 min at 15,000 Â g to
remove large aggregates, which is the main source of noise. Always
prepare volumes of at least 20 μl of sample in the smallest microreaction tubes to avoid surface absorption.
3.2.2 Fluorescence
Pretests
Before starting binding assays, several pretests are required to optimize. Check the optimal concentration of the fluorescence molecules. Ideal fluorescent intensities are between 200 and 1500
counts.
Check the capillary type that is optimal for our study (standards, hydrophobic, and premium). The aim is to obtain narrow,
shoulder-free, regular, and symmetrical fluorescent peaks.
Check the optimal buffer composition. BSA and detergents can
be used to improve sample homogeneity. pH and/or ionic strength
are also parameters that can be adjusted if needed. Reducing agents
freshly prepared can also be used when needed.
3.2.3 Preliminary Binding
Assays
In these preliminary assays, we compare the fluorescent signals of
the receptor in the absence and in the presence of the higher
concentration used for the ligand. The DNA solution (10 μl of
[DNA-FAM5] at 10 nM) is mixed with 10 μl buffer or with 10 μl of
Ku at 266 nM concentration. We analyzed fluorescence intensity
and homogeneity, and the signal to noise ratio by repeating the
measurement four times. MST traces should be smooth, which will
indicate the absence of aggregation. The signal is defined as the
response amplitude (difference of fluorescence amplitude between
receptor alone and receptor–ligand complex) and the noise is the
standard deviation of errors between replicates. A signal-to-noise
ratio less than 5 suggests a signal too weak to measure a K d , a ratio
of 5 or more indicates a quite favorable case for a K d measurement
and a ratio higher than 12 reflects optimal conditions to measure a
K d with MST.
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
137
ligand binding should be at least 6‰ (e.g., a change from an
F norm value of 857‰ to 863‰). Both the noise and the amplitude
are displayed when reviewing the dose–response panel. In order to
measure the K d , we incubate our (fluorescently labeled) receptor, at
a concentration below the K d , with increasing concentrations of
non-labeled ligand. An easy way to do this is to perform a dilution
series, in the form of 1:1 dilutions. In the Monolith instruments,
we can use 16 concentrations; this will cover a concentration range
of 3 Â 10
4 (2
15
).
3.2.1 Sample
Preparation
Non-labeled Ku and DNA are prepared as described in Subheading
3.1.2. First, always spin down the stocks for 5 min at 15,000 Â g to
remove large aggregates, which is the main source of noise. Always
prepare volumes of at least 20 μl of sample in the smallest microreaction tubes to avoid surface absorption.
3.2.2 Fluorescence
Pretests
Before starting binding assays, several pretests are required to optimize. Check the optimal concentration of the fluorescence molecules. Ideal fluorescent intensities are between 200 and 1500
counts.
Check the capillary type that is optimal for our study (standards, hydrophobic, and premium). The aim is to obtain narrow,
shoulder-free, regular, and symmetrical fluorescent peaks.
Check the optimal buffer composition. BSA and detergents can
be used to improve sample homogeneity. pH and/or ionic strength
are also parameters that can be adjusted if needed. Reducing agents
freshly prepared can also be used when needed.
3.2.3 Preliminary Binding
Assays
In these preliminary assays, we compare the fluorescent signals of
the receptor in the absence and in the presence of the higher
concentration used for the ligand. The DNA solution (10 μl of
[DNA-FAM5] at 10 nM) is mixed with 10 μl buffer or with 10 μl of
Ku at 266 nM concentration. We analyzed fluorescence intensity
and homogeneity, and the signal to noise ratio by repeating the
measurement four times. MST traces should be smooth, which will
indicate the absence of aggregation. The signal is defined as the
response amplitude (difference of fluorescence amplitude between
receptor alone and receptor–ligand complex) and the noise is the
standard deviation of errors between replicates. A signal-to-noise
ratio less than 5 suggests a signal too weak to measure a K d , a ratio
of 5 or more indicates a quite favorable case for a K d measurement
and a ratio higher than 12 reflects optimal conditions to measure a
K d with MST.
Measurements of Protein–DNA Complexes Interactions by Isothermal. . .
137