Centrifugal Protein Concentration and Buffer Exchange

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Centrifugal Protein Concentration/Buffer Exchange

  1. Obtain a centrifugal filter (Amicon or Centricon) with a molecular weight cutoff at least half of your protein’s weight.
  2. If the filter is new, run ~5 mL of nanopure water through before adding your protein mixture.
    1. Make sure to balance the centrifuge with another centrifugal filter. I typically use an old one. Regular falcon tubes will unbalance the centrifuge even though they are the same weight.
  3. Add your protein mixture up to the recommended line.
  4. Spin at 4,000 rpm for ~40 minutes.
  5. Pour out the filtrate. The supernatant contains your protein.
  6. Add the rest of your protein mixture or add in the new buffer.
  7. Continue to add and spin down the new buffer until it is sufficiently exchanged and the protein is concentrated.
    1. ~50 mL works well
    2. Be sure not to over concentrate and aggregate your protein.
  8. Keep the filter wet with 20% ethanol when it is stored.
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