Nov 17, 2015
Purification of viruses from culture lysates
- Janice E. Lawrence and Grieg F. Steward1
- 1Manual of Aquatic Viral Ecology
- VERVE Net
External link: http://www.aslo.org/books/mave/MAVE_166.pdf
Protocol Citation: Janice E. Lawrence and Grieg F. Steward 2015. Purification of viruses from culture lysates. protocols.io https://dx.doi.org/10.17504/protocols.io.d2t8em
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 20, 2015
Last Modified: November 24, 2017
Protocol Integer ID: 1843
Abstract
Ultracentrifugation provides a means to concentrate, analyze, and purify viruses in solution, and therefore represents an invaluable tool for aquatic virologists. This protocol provides a method for purification of viruses from culture lysates from natural water samples.
Guidelines
Materials and reagents
• OptiPrep (60% iodixanol solution)—Axis-Shield, Accurate Chemical and Scientific (Westbury), Progen Biotechnik, or Sigma Aldrich
• Open-topped ultracentrifuge tubes—i.e., Beckman Coulter Ultra-Clear
• Ultracentrifuge.
• Swing-out Ultracentrifuge Rotor—i.e., Beckman Coulter SW41, SW28, or MLS50
• 30 kDa cutoff disposable centrifugal ultrafiltration devices—i.e., Millipore
• 3-mL syringe with Luer-Lok or Luer-Slip
• Pipetting needle—i.e., Cadence Science, stainless-steel 14- or 16-guage 4-inch cannula with Luer hub or Slip hub
• Sterile 1.5 mL microcentrifuge tubes for collecting gradient fractions
• Sterile disposable transfer pipettes
• Sterile virus-free media for resuspending and diluting virus
• Polyethylene glycol, average molecular weight 6000- 8000—i.e., Fisher Scientific Carbowax PEG 8000, or Sigma Aldrich Biochemika Ultra 8000
Discussion
OptiPrep must be removed from samples before examination of virus particles by negative staining and TEM. This can be achieved using disposable Millipore centrifugal ultrafiltration devices with a 30 kDa cutoff. For most other applications OptiPrep does not need to be removed prior to further analysis, although it should be assayed to determine effects on the growth of specific viral-hosts when re-infection assays are used to confirm purification of the infectious agent.
Clarify lysate
Clarify lysate
Centrifuge the lysate at 4000g for 30 min.
00:30:00
Carefully decant and retain the supernatant.
Concentrate virus by PEG precipitation
Concentrate virus by PEG precipitation
Dissolve 8% PEG (w/v) in clarified lysate and allow to precipitate overnight at 4°C.
18:00:00
Centrifuge the PEG solution at 10,000g for 20 min.
00:20:00
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.
18:00:00
Concentrate sample to ~1 mL through a 30 kDa cutoff disposable centrifugal ultrafiltration device.
Prepare continuous, isopycnic, purifying gradients
Prepare continuous, isopycnic, purifying gradients
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.
02:00:00
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.
Collect viral fraction
Collect viral fraction
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).