concentration should be 10–20 times that of the K d . Quantities
listed below are required to characterize the Ku–DNA complex (the
expected K d is in the nM range).
For experiments with labeled DNA and non-labeled Ku, we
use:
1. Ku heterodimer purified as described in Subheading 2.2. A
total volume of 20 μl at the higher concentration is needed
per run.
2. A dialysis membrane with a molecular weight cut-off (MWCO)
of 30 kDa (Spectra/Por
® molecular porous membrane tubing); 2 l of MST buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 10 mM MgCl 2 , 0.05% Tween-20).
3. Desalted 18 bp or 42 bp oligonucleotides labeled with a
5
0 -FAM and their complementary strands synthetized at the
1 μmole scale (see sequences above with a FAM fluorophore
added in 5
0 ) (Eurofins). One synthesis yielded about
100 nmoles of oligonucleotides, sufficient for 50 titrations
after hybridization.
For experiments with non-labeled DNA and labeled Ku, you
will need:
4. Ku heterodimer purified as described in Subheading 2.2. The
buffer of Ku is changed for a buffer that does not contain
primary amines, using the labeling buffer of the Monolith
Protein Labeling kit BLUE-NHS (Amine Reactive) from
Nanotemper (reference MO-L003), for which composition is
not provided, but the optimal pH is between 8 and 8.4. A
volume of 100 μl at 20 μM is required.
5. MST buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
10 mM MgCl 2 , 0.05% Tween-20).
6. A Nanotemper labeling kit containing fluorescence dyes that
are suited for MST measurements. The protocols are optimized
to ensure a good labeling, an efficient removal of free dye and
about a 1:1 ratio of labeled protein to dye. Two coupling
chemistries are available, one for primary amines (NHS, e.g.,
lysine labeling) and one for thiol groups (maleimide for cysteine labeling). Second-generation dyes are now available (TrisNTA) but only for the instrument with red LED. Proteins with
a molecular weight greater than 5 kDa can be labeled.
7. Desalted 18 bp or 42 bp non-labeled oligonucleotides and
their complementary strands are synthetized at the 1 μmole
scale. The DNA sequences are the same as indicated above.
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Amandine Gontier et al.
listed below are required to characterize the Ku–DNA complex (the
expected K d is in the nM range).
For experiments with labeled DNA and non-labeled Ku, we
use:
1. Ku heterodimer purified as described in Subheading 2.2. A
total volume of 20 μl at the higher concentration is needed
per run.
2. A dialysis membrane with a molecular weight cut-off (MWCO)
of 30 kDa (Spectra/Por
® molecular porous membrane tubing); 2 l of MST buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 10 mM MgCl 2 , 0.05% Tween-20).
3. Desalted 18 bp or 42 bp oligonucleotides labeled with a
5
0 -FAM and their complementary strands synthetized at the
1 μmole scale (see sequences above with a FAM fluorophore
added in 5
0 ) (Eurofins). One synthesis yielded about
100 nmoles of oligonucleotides, sufficient for 50 titrations
after hybridization.
For experiments with non-labeled DNA and labeled Ku, you
will need:
4. Ku heterodimer purified as described in Subheading 2.2. The
buffer of Ku is changed for a buffer that does not contain
primary amines, using the labeling buffer of the Monolith
Protein Labeling kit BLUE-NHS (Amine Reactive) from
Nanotemper (reference MO-L003), for which composition is
not provided, but the optimal pH is between 8 and 8.4. A
volume of 100 μl at 20 μM is required.
5. MST buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
10 mM MgCl 2 , 0.05% Tween-20).
6. A Nanotemper labeling kit containing fluorescence dyes that
are suited for MST measurements. The protocols are optimized
to ensure a good labeling, an efficient removal of free dye and
about a 1:1 ratio of labeled protein to dye. Two coupling
chemistries are available, one for primary amines (NHS, e.g.,
lysine labeling) and one for thiol groups (maleimide for cysteine labeling). Second-generation dyes are now available (TrisNTA) but only for the instrument with red LED. Proteins with
a molecular weight greater than 5 kDa can be labeled.
7. Desalted 18 bp or 42 bp non-labeled oligonucleotides and
their complementary strands are synthetized at the 1 μmole
scale. The DNA sequences are the same as indicated above.
132
Amandine Gontier et al.