Using the RFP-Trap Agarose Kit on Nemtostella vectensis embryos injected with a plasmid containing the fluorescent protein mCherry bound to the acidic coral protein CARP1.

For this extraction I used the manufacturer protocol with some adjustments.

Embryo cell lysis

  1. Use a tissue homogenizer to lyse the embryos inside a 2 mL cryo tube containing 200 µL Lysis buffer together with a protease inhibitor cocktail and 1 mM PMSF.
  2. Place the tube on ice for 30 minutes, pipetting the suspension every 10 min.
  3. Centrifuce the lysate at 17000g for 10 minutes at +4°C. Transfer the supernatant to a new tube and add 300 µL Dilution buffer together with a protease inhibitor cocktail and 1 mM PMSF.

Bead equilibration

  1. Resuspend the beads by gently pipetting up and down.
  2. Transfer 25 µL of bead slurry into a new 1.5 mL tube.
  3. Add 500 µL ice-cold Dilution buffer.
  4. Sediment the beads by centrifugation at 2500x g for 5 min at +4°C. Discard the supernatant.

Protein binding

  1. Add diluted lysate to the equilibrated beads.
  2. Rotate end-over-end for 1 hour at +4°C.

Washing

  1. Sediment the beads by centrifugation at 3000x g for 5 min at +4°C.
  2. Save the supernatant for further analysis (flow-through/non-bound fraction).
  3. Discard remaining supernatant.
  4. Resuspend beads in 500 µL Wash buffer.
  5. Sediment the beads by centrifugation at 3000x g for 5 min at +4°C. Discard remaining supernatant.
  6. Repeat this step at least twice.
  7. During the last washing step, transfer the beads to a new tube.

Elution with 2x SDS-sample buffer (Laemmi)

  1. Remove the remaining supernatant.
  2. Resuspend beads in 80 µL 2x SDS-sample buffer(not provided by the kit).
  3. Boil beads for 5 min at +95°C to dissociate protein complexes from the beads.
  4. Sediment the beads by centrifugation at 3000x g for 2 min at +4°C.
  5. Analyze the supernatant in SDS-PAGE. See the protcol here.