Desthiobiotin datp

25 μg of forward and reverse sequence oligonucleotides (Table 1) were diluted in annealing buffer (20 mM Tris–HCl, pH 7.5, 10 mM MgCl₂, 100 mM KCl), denatured at 95°C, and annealed by cooling. Annealed double‐strand oligonucleotides were incubated with 100 units T4 kinase (Life Technologies) for 2 h at 37°C followed by incubation with 20 units T4 ligase overnight. Concatenated DNA strands were purified using phenol–chloroform extraction. Following biotinylation with desthiobiotin‐dATP (Jena Bioscience) and 60 units DNA polymerase (Thermo Scientific), the biotinylated probes were purified using microspin G‐50 columns (GE Healthcare). Telomeric or control DNA was immobilized on 375 μg paramagnetic streptavidin beads (Dynabeads MyOne C1, Thermo Scientific) on a rotation wheel for 30 min at room temperature. Subsequently, baits were incubated with 40 μg nuclear extract in PBB buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 0.5% Igepal CA‐630 (Sigma) and 1 mM DTT) while rotating for 2 h at 4°C. 20 μg sheared salmon sperm DNA (Ambion) was added as a competitor for DNA binding. After three washes with PBB buffer, bound proteins were eluted in 2× Laemmli buffer (Sigma‐Aldrich), boiled for 5 min at 95°C and separated on a 4–12% NuPAGE Novex Bis–Tris precast gel (Thermo Scientific).

Forward and reverse sequence oligonucleotides (25 μg) (see Supplementary Table 3) were diluted in annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 100 mM KCl), denatured at 95 °C and annealed by cooling. Annealed double-stranded oligonucleotides were incubated with 100 units T4 kinase (Life Technologies) for 2 h at 37 °C followed by incubation with 20 units T4 ligase overnight. Concatenated DNA strands were purified using phenol–chloroform extraction. Following biotinylation with desthiobiotin-dATP (Jena Bioscience) and 60 units DNA polymerase (Thermo), the biotinylated probes were purified using microspin G-50 columns (GE Healthcare). Telomeric or control DNA was immobilized on 500 μg paramagnetic streptavidin beads (Dynabeads MyOne C1, Life Technologies) on a rotation wheel for 30 min at room temperature. Subsequently, baits were incubated with 400 or 800 μg (frog) of nuclear extract in PBB buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 0.5% Igepal CA-630 (Sigma)) while rotating for 1.5 h at 4 °C. Sheared salmon sperm DNA (10 μg; Ambion) was added as a competitor for DNA binding. After three washes with PBB buffer, bound proteins were eluted in 1 × LDS sample buffer supplemented with 0.1 M DTT, boiled for 10 min at 70 °C and separated on a 10% NuPAGE Novex Bis-Tris precast gel (Life Technologies).