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. Author manuscript; available in PMC: 2016 Jun 28.
Published in final edited form as: Methods Cell Biol. 2015 Sep 2;131:467–486. doi: 10.1016/bs.mcb.2015.06.010

Figure 2. Schematic workflow for RNA affinity chromatography, described in section 2.

Figure 2

First, the biotinylated RNA probe is prepared (section 2.2), preferably in advance. However the axonal extract should be prepared fresh each time (section 1.2.). The last centrifugation step of the axoplasm preparation can be used to equilibrate Streptavidin Dynabeads. Half of the beads will be used for the preclearing of the axoplasm (2.3.3), the other half will be linked to the RNA bait (2.3.2). Since both steps require incubation for 1h at 4°C, this can be performed in parallel. Afterwards, the axoplasm will be transferred to the conjugated beads and incubated for at least 2h to o/n at 4°C with overhead rotation (2.3.4). After 3 washes in high salt buffer proteins are eluted using RNase A and the sample is prepared for MS (2.3.5).