Figure 5: Pull-down experiments.

(a) Schematic representation of the fusion proteins. (b) Protocol for the pull-down experiment. (c) Purified Ops-FG-SNAP and Ops-FG proteins are shown in the SDS–PAGE/Coomassie-stained gel panels labelled ‘Purified proteins’ (5 μl samples were loaded from ∼100 ng μl⁻¹ stocks; it is noteworthy that the Ops-FG-SNAP constructs stain more intensely than the Ops-FG proteins). The pull-down experiment was carried out as described in ‘Methods’ and as outlined in b. Ops-FG-SNAP protein (2 μg) corresponding to either WT or RP-associated mutants was covalently pre-bound to SNAP capture resin in 0.1% (w/v) DDM and the resin was washed before adding cognate Ops-FG protein (2 μg in 0.1% (w/v) DDM). The mixture was treated with Biobeads as described in ‘Methods’ before removing the supernatant, washing the resin and analysing the resulting pulldowns by SDS–PAGE/Coomassie staining after elution from the resin with Laemmli buffer (panel labelled ‘Pull-down’). The pull-down gel is representative of four independent experiments. Entire gels corresponding to the panels ‘Purified proteins’ and ‘Pull-down’ are shown in Supplementary Fig. 6C,D.