WinterVomitingLab:Protocols/Amicon ultrafiltration

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Overview

Concentration of human or animal caliciviruses using Amicon (Millipore) centrifugal ultrafilters. Procedure can also be used for buffer exchange.

Materials

  • Amicon (Millipore) centrifugal ultrafilters
    • 50K or 100K MWCO
    • Sizes range from 100 ul to 50 ml :*[| see example]
  • 3% beef extract or bovine serum albumin (optional)
  • Virus stock
  • Exchange buffer (if desired)
  • extra-long 200 ul pipette tips

Procedure

Pre-treatment of filters

  1. Add 3 ml of filtered sterile DI water in the top part of Amicon ultrafilters (we use 50K or 100K MWCO filters).
  2. Centrifuge at 3,000 x g for 1-2 minutes, until the liquid has passed through the membrane.
  3. Discard the filtrate (in the bottom part).
 * Note: an additional pre-treatment of the filtres with 3% beef extract or bovine serum albumin 
   can improve virus recovery; however, it is not suitable for all applications as it is retained 
   using filters ≥ 30 MWCO.  If your goal is to purify a virus stock, do not perform pre-treatment 
   with BSA.

Concentration of virus

  1. Add 3-4 ml of sample in the top part of the filter.
  2. Centrifuge at 3,000 x g until the volume is ~ 0.2 ml. To do this,
    1. Spin for 5 minutes and check retentate volume
    2. Spin an additional 5 minute and check again
    3. Repeat until ~0.2 – 0.25 ml are remaining (do not let volume reduce below 0.2 ml. This can cause aggregation.)
  3. Discard the filtrate
  4. Bring volume of retentate up to the desired volume of virus stock.
  5. Recover virus from the membrane, use a 200 µl pipette tip and insert the tip in the bottom of the filter unit. Withdraw the sample in several aliquots, careful not to puncture the membrane. It is important to recover the virus from the filter immediately after final centrifugation to maximize recovery of the virus. You will need to use the extra long 200 ul pipet tips for this step.

Buffer exchange

  1. After concentrating the virus stock as above, bring the sample volume up to 4 ml using the desired buffer.
  2. Centrifuge as before, leaving ~0.2 ml of retentate.
  3. Discard filtrate.
  4. Repeat the steps above until your sample has become sufficiently dilute, reloading 4 ml of the desired buffer before each centrifugation.
  5. Recover virus from the membrane as described above.

Notes

Some helpful tips to keep in mind

  1. Do not let the filters dry out. May result in poor recovery. Once, you add your first liquid, keep it wet until the very end when you recover your virus.
  2. Do not let the retentate volume reduce below 0.2 ml. This can cause aggregation.
  3. Don’t over-concentrate. Over-concentration can lead to precipitation and potential sample loss.
  4. It is important to recover the virus from the filter immediately after final centrifugation to maximize recovery of the virus.
  5. Do not exceed the device capacity of ~2 x 10^8 infectious virus particles.
  6. If samples passes through membrane, use a smaller MWCO filter
 * Filters are not compatible with the following:
   * Choroform
   * PEG concentrations  >10%
   * ethanol concentrations >70%
   * detergent (Tween, SDS) concentrations >0.1%
   * For a complete list of chemical compatibility, see User’s Guide.


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

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books