Tnt in vitro transcription translation system

The coding region of WRKY10 with an added MYC tag was inserted into the XhoI and KpnI sites of the pTNT vector. The coding region of WRKY2 was cloned into the TOPO vector and was then recombined into pDEST17. Both proteins were synthesized in the TnT in vitro transcription/translation system (Promega). The coding region of CCA1 or LHY was inserted into the XhoI and KpnI sites of the pMAL-c5x vector. The two MBP-tagged proteins were produced in E. coli host-strain BL21 (DE3) cells. Half a liter of culture was used for protein purification, and the recombinant proteins were eluted in a final volume of 2 ml. The DNA probes were synthesized with oligonucleotides that contained the putative DNA-binding elements and labeled with biotin. Binding reactions contained 10 mM Tris–HCl (pH 7.5), 150 mM KCl, 1 mM DTT, 2.5% (v/v) glycerol, 0.05 μg/μl poly (dI⋅dC), 1 μM ZnCl₂, and 0.05% NP-40. Reactions were incubated on ice for 30 min and loaded on native 5% polyacrylamide gels in 0.5× TBE buffer. Gels were run in 0.5× TBE buffer and electro-blotted to Hybond-N⁺ nylon membrane (Amersham). Detection of the biotin-labeled complex was performed using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) according to the manufacturer’s protocol. Increasing amounts of non-labeled oligonucleotides were used in the competition experiments.