WinterVomitingLab:Protocols/PEG precipitation

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Contents

Overview

Non-enveloped virus concentration using Polyethylene Glycol (PEG) precipitation.

Materials

  • Polyethylene Glycol with MW of 6,000 to 10,000
  • NaCl
  • Beef extract or Bovine serum albumin (optional)
  • 0.1 M PBS

Procedure

  1. Large particulates in aqueous samples should first be removed by centrifugation or filtration. Particulates may contain viruses. Consider organic solvent extraction of particulates. Supernatants derived from extraction of the particulates can be added to the aqueous volume previously removed.
  2. Adjust the pH to 7.0-7.2.
  3. Add 8% PEG (w/v). Use high molecular weight PEG of at least 6,000. MWs of 8,000 or 10,000 may enhance recovery.
  4. Add 0.3 M NaCl.
  5. If the sample is free of turbidity/small particulates, adding 3% beef extract or Bovine Serum Albumin (BSA) can enhance virus precipitation.
  6. Dissolve PEG by stirring or shaking at 4°C for a minimum of 4 hr. Dissolving for longer time periods (overnight) may enhance recovery.
  7. Centrifuge samples at 4°C for 30 min at a minimum speed of 9,000 x g.
  8. Decant supernatant as completely as possible without disturbing the pellet.
  9. If there is a small volume (less than 1-5 ml) of supernatant remaining in the sample, it can be used to re-suspend the pellet. If the volume is less than 1 ml, 0.1 M PBS can be added to re-suspend the pellet to the desired volume.
  10. Pipette up and down to thoroughly suspend the pellet. If no pellet can be visualized, pipette up and down in the area (and just beyond) where the pellet would form.
  11. Collect the precipitated virus sample. Note: PEG will be pelleted with the virus. There may also be other proteins/particulates co-precipitated. It may be necessary to perform a virus purification step depending on downstream applications.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
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  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books


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