Purification of viruses from culture lysates
Centrifuge the lysate at 4000g for 30 min.
Carefully decant and retain the supernatant.
Dissolve 8% PEG (w/v) in clarified lysate and allow to precipitate overnight at 4°C.
Centrifuge the PEG solution at 10,000g for 20 min.
Carefully decant the supernatant, retaining the pellet.
Resuspend pellet in a small volume of residual PEG solution and pool all pelleted material.
Repeat steps 5-6 as needed to concentrate virus to < 1 mL.
Resuspend virus in 10-50 volumes of culture media to dilute PEG and allow virus pellet to disaggregate overnight at 4°C.
Concentrate sample to ~1 mL through a 30 kDa cutoff disposable centrifugal ultrafiltration device.
Prepare OptiPrep solutions using culture media as the diluent.
Using the underlayering technique with syringe and pipetting needle, pour 4-step gradients into open-toped ultracentrifuge tubes, beginning with the least dense solution first.
Allow to blend for 2 h at room temperature.
Mark the top of the gradients with a fine-tipped marker, and carefully overlay virus concentrate using a transfer pipette.
Overlay culture media on balance gradients to create balance tubes.
Balance the tubes by adding media to underweight tubes.
Load tubes into rotor and ultracentrifuge at maximum permissible speed until density equilibrium is reached.
Using any fraction collection apparatus/technique, carefully extract purified viral concentrate from each tube.
If bands are not visible, a starting point is to fractionate the gradient into 4 + fractions, and use a couple of techniques to identify the virus-containing fraction (i.e., TEM, bioassay, absorbance at 260 nm, nucleic acid analysis, epifluorescence microscopy or flow cytometry).
