3.2.4 K d Determination
Prepare 16 small micro-tubes and transfer 20 μl of the highest
concentration of the ligand (non-labeled molecule) to the first
one. The Concentration Finder software tool can be used to simulate the interaction with different concentrations of ligand and
receptor. Fill 10 μl of the optimal assay buffers into the tubes
2–16. Use the same pipette tip and transfer 10 μl of tube 1 to
tube 2 and mix well by pipetting up and down several times. Do the
same with tube 3 to tube 16 to obtain a serial dilution. Remove
10 μl from tube 16 after mixing. Add 10 μl of receptor (labeled
molecule) to each tube and mix well by pipetting up and down
several times. Here change tip for every well to ensure the addition
of the same quantity in each tube and no contamination of the
labeled molecule that may be due to the pipetting. Incubate if
needed before loading into the capillaries. Most interactions reach
equilibrium after a few minutes, so additional incubation is often
not needed. When larger conformational changes are required from
both partners, slow kinetics can be observed, and longer incubations might be necessary. Fill 16 capillaries of the correct type and
place them in the instrument. Start the measurements.
3.2.5 Assays
with Labeled Ku (and
Non-labeled DNA)
Ku is labeled with NT495 final concentration 20 nM. Titrant DNA
18 bp is prepared at concentrations ranging from 123 nM to 3.9
pM by serial dilution. The buffer used is MST buffer and the
capillaries are premium capillaries. Measurements are performed
at 22
C, at 40% LED excitation and 40% or 60% MST power.
The signal to noise ratio is 3 in these conditions and does not enable
to measure a reliable K d (Fig. 4a). The excitation power of 40% was
enough to have around 350 fluorescence counts. Increasing the
MST power increases the signal, but also the noise of the data.
3.2.6 Assays
with Labeled DNA (and
Non-labeled Ku)
DNA is purchased with a FAM fluorophore in 5
0 of one 18 nt
single-strand DNA. The fluorescent probe is separated to the
18 nt by three nucleotides to avoid interference with the binding
site (Sigma Aldrich, HPLC 95% pure). The stock DNA is prepared
at 230 μM with the same hybridization protocol as for ITC (see
above). The final concentration of DNA in MST assays is 10 nM.
Ku is prepared by serial dilution from 1330 nM to 80 pM. The
buffer used is the MST buffer. Standard capillaries are used as well
as low binding tubes and tips. Measurements are performed at 60%
(data not shown), 80%, and 100% LED excitation, 40% MST
power, and temperature of 22
C (Fig. 4b, d). The signal to noise
ratio at 60%, 80%, and 100% are 5.8, 8.3, and 17.7, respectively.
The K d is poorly defined at 60% excitation power with a K d estimated of 100 Æ 500 nM (data not shown). We measured a more
precise K d of 16.0 Æ 7.7 nM at 80% (standard error of regression of
1.04 and a reduced χ
2 of 0.95) (Fig. 4b). At 100% excitation power,
we measured the K d with the smallest standard error, K d of
10.8 Æ 2.6 nM (standard error of regression is 0.37 and reduced
χ
2 is 0.19) (Fig. 4c, d).
138
Amandine Gontier et al.
Prepare 16 small micro-tubes and transfer 20 μl of the highest
concentration of the ligand (non-labeled molecule) to the first
one. The Concentration Finder software tool can be used to simulate the interaction with different concentrations of ligand and
receptor. Fill 10 μl of the optimal assay buffers into the tubes
2–16. Use the same pipette tip and transfer 10 μl of tube 1 to
tube 2 and mix well by pipetting up and down several times. Do the
same with tube 3 to tube 16 to obtain a serial dilution. Remove
10 μl from tube 16 after mixing. Add 10 μl of receptor (labeled
molecule) to each tube and mix well by pipetting up and down
several times. Here change tip for every well to ensure the addition
of the same quantity in each tube and no contamination of the
labeled molecule that may be due to the pipetting. Incubate if
needed before loading into the capillaries. Most interactions reach
equilibrium after a few minutes, so additional incubation is often
not needed. When larger conformational changes are required from
both partners, slow kinetics can be observed, and longer incubations might be necessary. Fill 16 capillaries of the correct type and
place them in the instrument. Start the measurements.
3.2.5 Assays
with Labeled Ku (and
Non-labeled DNA)
Ku is labeled with NT495 final concentration 20 nM. Titrant DNA
18 bp is prepared at concentrations ranging from 123 nM to 3.9
pM by serial dilution. The buffer used is MST buffer and the
capillaries are premium capillaries. Measurements are performed
at 22
C, at 40% LED excitation and 40% or 60% MST power.
The signal to noise ratio is 3 in these conditions and does not enable
to measure a reliable K d (Fig. 4a). The excitation power of 40% was
enough to have around 350 fluorescence counts. Increasing the
MST power increases the signal, but also the noise of the data.
3.2.6 Assays
with Labeled DNA (and
Non-labeled Ku)
DNA is purchased with a FAM fluorophore in 5
0 of one 18 nt
single-strand DNA. The fluorescent probe is separated to the
18 nt by three nucleotides to avoid interference with the binding
site (Sigma Aldrich, HPLC 95% pure). The stock DNA is prepared
at 230 μM with the same hybridization protocol as for ITC (see
above). The final concentration of DNA in MST assays is 10 nM.
Ku is prepared by serial dilution from 1330 nM to 80 pM. The
buffer used is the MST buffer. Standard capillaries are used as well
as low binding tubes and tips. Measurements are performed at 60%
(data not shown), 80%, and 100% LED excitation, 40% MST
power, and temperature of 22
C (Fig. 4b, d). The signal to noise
ratio at 60%, 80%, and 100% are 5.8, 8.3, and 17.7, respectively.
The K d is poorly defined at 60% excitation power with a K d estimated of 100 Æ 500 nM (data not shown). We measured a more
precise K d of 16.0 Æ 7.7 nM at 80% (standard error of regression of
1.04 and a reduced χ
2 of 0.95) (Fig. 4b). At 100% excitation power,
we measured the K d with the smallest standard error, K d of
10.8 Æ 2.6 nM (standard error of regression is 0.37 and reduced
χ
2 is 0.19) (Fig. 4c, d).
138
Amandine Gontier et al.