Fig. 2.

Csy4-based pull-down strategy using a conditional endoribonuclease. (A) Scheme of Csy4-based RNA affinity purification. Avidin agarose, biotinylated Csy4*, and Csy4 hairpin-tagged transcript are preincubated to make a ternary complex, which is then incubated with cell extract. After washing, the RNA–protein complexes are eluted by cleaving off the Csy4 hairpin tag and releasing RNA–protein complexes on the addition of imidazole. (B) Efficient RNA recovery of Csy4-based RNA purification with HeLa cell extract. Tagged RNA transcripts, biotinylated Csy4*, and avidin agarose resin were incubated for 2 h at 4 °C and subjected to mock purification. The samples were separated on 12% denaturing PAGE and visualized by staining with SYBR Gold (Invitrogen).