Which methods can I combine for automated two step purification?

A Two Step purification system can easily be used to combine two methods of your purification strategy. A classical approach is the combination of a capture with a polishing step. Some examples for the first and the second step are listed in the table below. In principle any method can be combined. Please consider while designing your two step strategy that the elution conditions of the first step should match the starting conditions of the second step. A typical example for two step purification is the purification of His-tagged proteins. The capture step for these proteins is done by immobilized metal ion affinity chromatography. The elution is achieved by imidazole. The next logical step which can be easily integrated in a two step set up is a desalting column to exchange the buffer. Hereby the imidazole is removed, and the ionic strength can be reduced.






  • Tag-affinity (e.g. GST, Strep-Tag)
  • Antibody affinity (e.g. Protein A, Protein G)
  • Immobilized metal ion affinity (IMAC) (e.g. Ni-NTA, Talon)
  • Ion exchange chromatography

Intermediate/ Polishing

  • Size exclusion chromatography
  • Desalting/buffer exchange
  • Ion exchange chromatography
  • Hydrophobic interaction chromatography

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