RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction

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.  

Pre-wash 50 microliters of streptavidin-magnetic beads per 50 picomoles of RNA. Vortex the tube with streptavidin-magnetic beads for 15 seconds. After vortexing quickly, remove 200 microliters into a clean 1.5-milliliter safe lock tube using cut pipette tips. Next, place the tube on a magnetic stand so that the beads collect at the side of the tube, and wait one minute. Remove the resuspension liquid.

To wash the beads, remove the tube from the magnetic stand at 400 microliters of 0.1 molar sodium hydroxide, 0.05 molar sodium chloride solution, and gently pipette up and down several times. Place the tube back on the magnetic stand and wait one minute, then, collect the supernatant.

Wash the beads with 200 microliters of 100-millimolar sodium chloride, and remove the supernatant. Add 200 microliters of 20 millimolar Tris. Resuspend the beads by pipetting and place the tube on the magnetic stand. After one minute, remove the supernatant. Then, remove the tube from the magnetic stand at 200 microliters of 1x RNA capture buffer and resuspend streptavidin-magnetic beads by briefly vortexing.

Remove 50 microliters of streptavidin-magnetic beads and add it to each labeled-RNA tube using a cut pipette tip. Incubate the tubes for 30 minutes at room temperature on a roller. After the tube has been on the magnetic stand for one minute, remove the supernatant. Add 50 microliters of 20 millimolar Tris to the beads and resuspend them. Then, place the tubes into the magnetic stand. After one minute, remove the supernatant.

Add 100 microliters of 1x protein-RNA binding buffer to the beads and resuspend them. Then, place the tubes back into the magnetic stand. Once the tubes have been in the stand for 1 minute, collect the supernatant. Add 100 microliter of master mix B. Resuspend by gently pipetting up and down and avoid creating bubbles. Incubate reaction tubes for 60 minutes at 4 degrees Celsius on a roller.

Move on to the wash and elution steps. Place the tubes into a magnetic stand. Collect the flow-through and transfer it into a new tube. Pipette 100 microliters of 1X wash buffer on the beads. Resuspend gently. Place them back into the stand. Wait one minute and discard the supernatant. Repeat the step two times.

Add 40 microliters of elution buffer to the magnetic beads. Mix by pipetting up and down and incubate on a tube shaker. Place the tubes into a magnetic stand, wait one minute, and collect eluate sample. Place on ice and use for downstream analysis.