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Protein PurificationExtraction and Clarification

Removal of DNA

Most lysis methods cause the release of nucleic acids (DNA and RNA). These have to be removed because they can cause viscosity problems or due to their interference with subsequent chromatographic steps. Different methods exist:

  • Enzymatic digestion by the addition of DNase I (1 µg/ml) to the cell lysate. The mixture is incubated on ice for 10-15 min.
  • Mechanical breakdown by shearing during sonication. When the French Pressure Cell is used it is advisable to add DNase to the cell suspension.
  • Precipitation by treatment with polyethyleneimine (0.1% (w/v)) or protamine sulphate (1% (w/v)) followed by centrifugation. Add the precipitants to the cell lysate and incubate the solution for 30 min at 4°C.