Ultraviolet crosslinker purple

First, biotin label was linked to the 3′ end of artificially synthesized single-stranded oligonucleotide probe containing binding site by DNA 3′ end biotin label kit (Beyotime, Shanghai, China). Second, double-stranded DNA probe with biotin label was obtained by annealing with artificially synthesized complimentary chain. Third, purified CkREV protein was incubated with probe with biotin label at a certain proportion while unlabeled double-stranded probe was used as cold probe. Fourth, native-PAGE was employed to separate samples before being transferred onto nylon membrane (Solarbio, Beijing, China) with positive charge through wet transformation method. Fifth, the nylon membrane was placed under ultraviolet crosslinker purple (UVP, Upland, USA) at 254 nm, 120 mJ/cm² for 60 s. Last, colour development was employed on a completely cross-linked nylon membrane by EMSA chemiluminescence kit (Beyotime, Shanghai, China) for observation under chemiluminescence imager. Detailed steps complied with instructions of EMSA chemiluminescence kit (Beyotime, Shanghai, China).