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. Author manuscript; available in PMC: 2024 Jun 3.
Published in final edited form as: Methods. 2016 Nov 18;118-119:41–49. doi: 10.1016/j.ymeth.2016.11.009

Fig. 2.

Fig. 2.

Illustration of steps during PAR-CLIP. (A) Immunoblot using antibody against the YBX1 protein. Samples from left to right: input, IP flow through, “off-beads” and “on-beads” IP elutions. (B) Autoradiograph showing the separation of radiolabeled YBX1 RNP immunoprecipitated from “off-beads” and “on-beads” procedures. Eluted RNPs are separated by SDS-PAGE and transferred to a nitrocellulose membrane. (C-E) Autoradiography of denaturing polyacrylamide gels that are used along the “off-beads” procedure to visualize and select: (C) RNA fragments extracted after Proteinase K digestion of RNPs, (D) products of 3′ adapter ligation, and (E) products of 5′ adapter ligation. (F, G) Agarose gel separation of PCR products at increasing cycle numbers for off-beads (F) and on-beads (G) procedures. For library preparation we chose 15 and 18 cycles, respectively. The expected PCR product runs at ~100 bp. Linker-linker side-products run at 71 bp.