Editing Qbeta VLP (section)

== Purification of Qβ ==

<br/>1. Resuspend the cell pellet with ~20–30 mL of 0.1 M potassium phosphate buffer (pH 7.00).<br/>2. Make sure the resuspension has no chunks, and lyse the cells using a microfluidizer processor according to manufacturer’s protocol (see<br/>Table of Materials). Lyse the cells at least twice to increase the yield of the particles.<br/>3. Centrifuge the lysate in 250 mL centrifuge bottles at 20,621 x g for 1 hr at 4 °C.<br/>4. Discard the pellet and measure the volume of supernatant in mL. Multiply that value by 0.265 and add that amount of g of ammonium sulfate<br/>to the supernatant.<br/>5. Stir at 4 °C for at least 1 h on a stir plate at 200 rpm to precipitate out the protein.<br/>6. Centrifuge in 250 mL bottles at 20,621 x g for 1 h at 4 °C.<br/>7. Discard supernatant and resuspend the pellet with about 10 mL of 0.1 M potassium phosphate buffer (pH 7.00).<br/>8. Add equal volumes of 1:1 chloroform/n-butanol to the crude sample and mix by vortexing for a few seconds.<br/>9. Centrifuge in 38 mL tubes at 20,621 × g for 30 minutes at 4 °C.

10. Recover the top aqueous layer using a Pasteur pipette. Be cautious not to take any of the gel like layer that has formed between the aqueous<br/>and organic layer.<br/>11. Thaw six 5–40% pre-made sucrose gradients and load about 2 mL of the extract onto each.<br/>12. Ultracentrifuge at 99,582 x g for 16 h at 4 °C with free deceleration.<br/>13. Shine a light emitting diode (LED) light under each tube and a blue band should become visible. Recover these particles with a long needle<br/>syringe.<br/>14. Ultrapellet the particles at 370,541 x g for 2.5 h at 4 °C.<br/>15. Discard the supernatant and resuspend the transparent pellet of purified particles with 0.1 M potassium phosphate buffer (pH 7.00).