Revision as of 11:32, 13 September 2012

Dialysis of the VLPs

Reagents:

Materials:

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.

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

@@ Line 6: / Line 6: @@
 <ul>
 <li>Formation Buffer</li>
-<li>Formation + 8M urea</li>
+<li>Formation Buffer + 8M urea</li>
 </ul>