Team:Wageningen UR/Protocol/DialysisPolero

From 2012.igem.org

Dialysis of the VLPs

This protocol is based on the CCMV VLP formation protocol. We made adjustments because Polero has a different pH at which the particle is stable. Besides, the number of dialysis steps is reduced and only 1 buffer is needed for the dialysis.

Reagents & Materials

Reagents:

  • Formation Buffer
  • Formation Buffer + 8M urea

Materials:

  • Pipettes + pipettepoints
  • Greinertubes
  • Dialysis tubing
  • Vortex
  • French Press
  • Centrifuge for Greiner tubes
  • Sorval or a similar centrifuge
  • Coldroom

Procedure

The temperature of the VLP’s is very important. All the work should be done in the cold room (4°C) and the used tubes have to be on ice.

  1. Resuspend the cells in 5 mL of Formation buffer
  2. Disrupt the cells by french press, cell pressure: 1000
  3. Add Formation buffer to 25 mL total volume
  4. Centrifuge the lysate until the insoluble fraction is pelleted – 4°C, 4700 rpm, 18 min
  5. Remove the supernatant completely and dissolve the pellet in 10 mL of cold Formation buffer containing 8 M urea (about 10-15 min). Vortex the pellet to easily resuspend it while cooling on ice. Do not allow the buffer to heat up above 25°C
  6. Allow the pellet to dissolve for 2-5 minutes
  7. Dilute the 10 mL of 8 M urea/ Formation buffer with 15 mL Formation buffer. If crystals form, the batch should be discarded
  8. Pellet everything that is not dissolved by centrifuging at 15000 rpm at 4°C for 15 minutes (Sorvall ultracentrifuge in the basement)
  9. Transfer the supernatant to a clean 50 mL Greiner tube
  10. Prepare a piece of dialysis tubing with a large diameter by soaking it in cooking demiwater until it opens up
  11. Put a tight knot on one side and fill the tubing with the supernatant
  12. Knot the tube on the other side leaving a small air bubble inside
  13. Dialyse the tube (about 25 mL volume) against 1x Formation buffer for 4 hours or overnight
  14. Dialysis is performed 6 times against 1x Formation(4h per dialysis, or overnight)


Next: Polero Purification