RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction
Transcript
To isolate RNA-binding proteins, or RBPs — post-transcriptional regulators — take a suspension of small RNA probes containing an adenylate-uridylate-rich element, or ARE — a sequence recognized by the target RBPs. The RNA probes are labeled with desthiobiotin — a biotin derivative.
Add magnetic beads coated with streptavidin — a protein with an affinity for biotin. Incubate under agitation to prevent the beads from settling. Desthiobiotin-labeled RNA binds to streptavidin, thus immobilizing the RNA on the beads.
Under the magnetic field, the beads collect on the side of the tube. Remove the supernatant. Resuspend the beads in a buffer providing an optimum pH for RBP-RNA binding.
Upon exposure to the magnet, beads collect on the side of the tube. Discard the supernatant. Resuspend the beads in a solution containing the target RBPs and incubate under agitation.
The target RBPs possess RNA recognition motifs that recognize the ARE sequence and bind to the RNA, thus forming an RNA-protein complex immobilized on the beads.
Under the magnetic field, the beads move toward the magnet without disturbing the protein-RNA interactions. Discard the supernatant. Resuspend the beads in an elution buffer containing biotin.
Biotin displaces the desthiobiotin from the beads by competitively binding to streptavidin, thus eluting the protein-RNA complex.
Assess the presence of the target RBPs in the eluate to confirm the protein-RNA interactions.
