3.1.2 Sample
Preparation for ITC
1. The Ku heterodimer (receptor) is dialyzed three times against
ITC buffer for at least 2 h against a volume of buffer
corresponding to at least 30-fold the volume of the receptor
sample. Keep 50 ml of the last dialysis bath as reference buffer.
It will be used to rinse the measurement cells and the syringe
and to adjust the concentration of the receptor and ligands, if
necessary.
2. Preparation of the receptor for the measurement cell: Concentrate the dialyzed protein using an Ultra-4 (Amicon), measure
UV absorption at 280 nm (using for example a Nanodrop;
Thermo Fisher Scientific), and adjust the concentration to
1 μM (0.15 mg/ml). A minimum volume of 2.3 ml (1.4 ml
for the measurement cell plus 0.9 ml dead volume) is prepared.
3. Hybridization of DNA substrates (ligand). The oligonucleotides are classically dissolved at 500 μM in water and equal
amounts of the complementary strands are mixed at a temperature of 95
C for 5 min and cooled down overnight at room
temperature. The concentration is estimated using a Nanodrop
(Thermo Fisher Scientific) and UV absorption at 260 nm.
4. Verify hybridization: Inject 6 nmoles (10 μl at 0,6 mM) on a
Mini-Q-1 ml (Sigma-Aldrich) column to check if the DNA is
correctly hybridized and the proportion of the remaining
single-strand DNA. Perform a gradient on the Mini-Q of
20 ml from 100% buffer A (Tris 20 mM, pH 8) to 100% buffer
B (Tris 20 mM, pH 8, NaCl 1 M).
5. The DNA ligand is diluted at 10 μM using the last dialysis
buffer to limit heat exchange due to buffer differences of the
receptor and the ligand. These dilution heats may mask the
desired signal. Prepare 0.6 ml of ligand to be positioned in the
serynge.
3.1.3 ITC Measurements
and Data Interpretation
1. The receptor (2.3 ml of Ku at 1 μM) and the ligand (0.6 ml
DNA at 10 μM) are degassed and equilibrated for 10 min at the
measurement temperature (here 20
C) using the
ThermoVac™.
2. The receptor is deposited in the measurement cell with a 2.5 ml
glass syringe. The sample is slowly loaded in the cell to avoid
bubbles. Injection is stopped when the liquid comes above a
visible level.
3. The ligand is loaded on the injection syringe (in open position)
using a 10-ml plastic syringe connected to the top of the ITC
syringe. The syringe is then purged and filled twice to remove
bubbles.
4. Start titration.
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Amandine Gontier et al.
Preparation for ITC
1. The Ku heterodimer (receptor) is dialyzed three times against
ITC buffer for at least 2 h against a volume of buffer
corresponding to at least 30-fold the volume of the receptor
sample. Keep 50 ml of the last dialysis bath as reference buffer.
It will be used to rinse the measurement cells and the syringe
and to adjust the concentration of the receptor and ligands, if
necessary.
2. Preparation of the receptor for the measurement cell: Concentrate the dialyzed protein using an Ultra-4 (Amicon), measure
UV absorption at 280 nm (using for example a Nanodrop;
Thermo Fisher Scientific), and adjust the concentration to
1 μM (0.15 mg/ml). A minimum volume of 2.3 ml (1.4 ml
for the measurement cell plus 0.9 ml dead volume) is prepared.
3. Hybridization of DNA substrates (ligand). The oligonucleotides are classically dissolved at 500 μM in water and equal
amounts of the complementary strands are mixed at a temperature of 95
C for 5 min and cooled down overnight at room
temperature. The concentration is estimated using a Nanodrop
(Thermo Fisher Scientific) and UV absorption at 260 nm.
4. Verify hybridization: Inject 6 nmoles (10 μl at 0,6 mM) on a
Mini-Q-1 ml (Sigma-Aldrich) column to check if the DNA is
correctly hybridized and the proportion of the remaining
single-strand DNA. Perform a gradient on the Mini-Q of
20 ml from 100% buffer A (Tris 20 mM, pH 8) to 100% buffer
B (Tris 20 mM, pH 8, NaCl 1 M).
5. The DNA ligand is diluted at 10 μM using the last dialysis
buffer to limit heat exchange due to buffer differences of the
receptor and the ligand. These dilution heats may mask the
desired signal. Prepare 0.6 ml of ligand to be positioned in the
serynge.
3.1.3 ITC Measurements
and Data Interpretation
1. The receptor (2.3 ml of Ku at 1 μM) and the ligand (0.6 ml
DNA at 10 μM) are degassed and equilibrated for 10 min at the
measurement temperature (here 20
C) using the
ThermoVac™.
2. The receptor is deposited in the measurement cell with a 2.5 ml
glass syringe. The sample is slowly loaded in the cell to avoid
bubbles. Injection is stopped when the liquid comes above a
visible level.
3. The ligand is loaded on the injection syringe (in open position)
using a 10-ml plastic syringe connected to the top of the ITC
syringe. The syringe is then purged and filled twice to remove
bubbles.
4. Start titration.
134
Amandine Gontier et al.