Dig gel shift kit

Fusion expression vectors, including MBP-PsTOE3, were constructed and used to transform E. coli BL21. The positive colonies were cultured at 37°C and 200 rpm until an OD₆₀₀ of 0.6–0.8 was obtained. Further, 0.1 mM IPTG (isopropyl β-d-thiogalactoside, isopropyl-β-d-thiogalactopyranoside) was added to induce the expression of fusion proteins; after this, the cells were cultured at 18°C and 150 rpm for 15 hours. The culture was centrifuged at 4000 rpm at 4°C for 15 minutes, and the cell pellet was suspended in binding buffer (20 mM Tris–HCl, 0.2 M NaCl, 1 mM EDTA, and 10 mM β-mercaptoethanol). The fusion proteins were purified from the inclusion bodies on Ni²⁺-NTA agarose resin (Qiagen) according to the manufacturer’s protocol.
The C-repeat (ACCGAC) was synthesized with four replications. The double-stranded probes were prepared by annealing and marked with the DIG Gel Shift Kit (Roche) with the final concentration of 0.155 μM. Poly(dI-dC) was used as a non-specific DNA competitor. EMSA was performed using a DIG Gel Shift Kit (Roche) according to the manufacturer’s instructions. As the negative control, the mutation C-repeat (AGCGAC) was synthesized with four replications. The primers used for EMSA are listed in Supplementary Data Table S1.

Single-stranded DNA oligonucleotides were labeled with DIG-11-ddUTP by terminal transferase using a Dig Gel Shift kit (Roche). The gel shift assays were performed using 1 μg of TcRPA-1 or increased concentrations (1, 2 and 4 μg) of TcRPA-2 or of each truncated mutants, and fixed concentrations of 0.155 pmol of labeled oligonucleotide, 0.1 μg of poly-L-lysine, 1 μg poly[d(I-C)], 4 μl of 5X binding buffer (100 mM HEPES pH 6.0, 5 mM EDTA, 50 mM (NH₄)₂SO₄, 5 mM DTT, Tween 20 1% (w/v), 150 mM KCl) in a final volume of 20 μl. The samples were maintained at room temperature for 15 min and then applied to a non-denaturing 6% gel (acrylamide/bis-acrylamide 37.5:1) run at 80 V in 0.25 X TBE buffer. Samples were then transferred onto a nylon membrane at 400 mA for 30 min in 0.25 X TBE and were fixed by UV light for 15 min. Detection of labeled oligonucleotides was performed using the Dig Gel Shift kit (Roche), according to the manufacturer's instructions.

Sense and antisense oligonucleotides, 5′-TAGAGACGGGG TTTCACCGTGTTACCAGG-3′ and 5′-GATCTAACACACGGTGAAACCCC GTCTCTATGC-3′, were annealed by stepwise cooling down from 95 °C to room temperature (RT) and were labeled with digoxigenin (DIG) using the DIG Gel Shift Kit (3353591910, Roche, Basel, Switzerland) to generate DIG-labeled probes for detecting Ku70 bound to DNA DSB^{99 (link)}. The DIG-labeled probe (0.8 ng) was incubated with 40 μg of GST-fusion proteins for 30 min at RT in 25 μL of binding buffer (1 μg of poly[d-(I-C)], 0.1 μg of poly-l-lysine, 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl₂, 0.5 mM DTT, 4% glycerol)l^{99 (link)}. The samples were separated by electrophoresis with 8% native polyacrylamide gel and 0.5× TBE buffer at 120 V for 1 h at 4 °C. After electrophoresis, the protein-probe complex and probe were transferred to Amersham Hybond-N+ membranes (RPN303B, Cytiva, Marlborough, MA, USA) for 30 min at 400 mA and cross-linked at 120 mJ for 3 min. The membrane was incubated with blocking buffer for 30 min, chemiluminescent detection buffer, and antibody Anti-Digoxigenin-AP antibody solution diluted 1 : 10,000 solution for 30 min following the protocol of DIG Gel Shift Kit (3353591910, Roche, Basel, Switzerlan). The signals were visualized by luminescent image analyzer (ImageQuant LAS 4000 mini, Cytiva, Marlborough, MA, USA).