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Latest revision as of 14:22, 24 October 2012

Dialysis of the VLPs

This protocol is based on the CCMV VLP formation protocol. We made adjustments because HepBcAg 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 Disassembly buffer
2. Disrupt the cells by french press, cell pressure: 1000
3. Add Disassembly 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 Disassembly buffer containing 8 M urea (about 10-15 min). Vortex the pellet to easily resuspend the 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/ Disassembly buffer with 15 mL Disassembly 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 Dialysis buffer for 4 hours or overnight
14. Dialysis is performed 6 times against 1x Formation(4h per dialysis, or overnight)

Next: HepB Purification