GFP pull down assay

Mix GFP-tagged TBK1 with 20µL of equilibrated GFP-Trap agarose beads (Chromotek) at a final concentration of 1micromolar (µM). Make sure to wash beads 2x in dH2O before washing with bead assay buffer to equilibrate the beads adding the protein to the beads.

To this end, wash 20µL of beads twice with dH₂O and equilibrate with bead assay buffer.

Resuspend the beads in 40µL bead assay buffer, to this add GFP-TBK1 at a final concentration of 5micromolar (µM).

Incubate the beads with GFP-TBK1 for 1h 0m 0s at 4°C at a horizontal tube roller.

Wash the beads three times to remove unbound GFP-tagged bait protein.

Prepare protein master mixes with prey protein in bead assay buffer at the following concentrations:

• mCherry-OPTN (1 µM),
• mCherry-NDP52 (1 µM),
• GST-NAP1 (1-10 µM).

Add the protein master mixes to the beads and incubate for 1h 0m 0s at 4°C at a horizontal tube roller.

Wash the beads three times to remove unbound proteins, remove any supernatant from the beads and resuspend the beads in 60µL of 1x Protein Loading dye, and heat-inactivate at 95°C for 0h 5m 0s.

Analyze the samples by SDS-PAGE and Coomassie staining as described above.