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Chloroform/Methanol Precipitation of Proteins


Here we present a method of concentrating proteins using chloroform and methanol which exploits phase separation of the two solvents to remove contaminating salts and detergents. The method is a two part process. Initially phase separation into hydrophobic and hydrophilic layers is induced by the addition of water and the idea is that proteins possessing both hydrophobic and hydrophilic layers will precipitate at the interface between the two layers. In the second part, the top aqueous layer is removed and protein is precipitated and pelleted.

NOTE: It is recommended to use ice-cold reagents when carrying out this protocol to encourage precipitation of the protein


  1. Pipette 100μl of protein sample into a 1.5 ml microcentrifuge tube
  2. Add 400 μl of methanol and vortex well
  3. Spin briefly to collect sample
  4. Add 100 μl of chloroform and vortex well
  5. Spin briefly to collect sample
  6. Add 300 μl of milliQ (or equivalent) grade water and vortex well (this step helps induce phase separation)
  7. Centrifuge at maximum speed (at least 13,000 rpm) for 2 mins
  8. Remove upper layer without disturbing the interface
  9. Add 300 μl of methanol and vortex well
  10. Centrifuge at maximum speed (at least 13,000 rpm) for 2 mins to pellet the protein
  11. Carefully remove the supernatent and leave the pellet to air-dry

Health and Safety

  • Methanol is highly flammable, keep away when using from sources of fire.
  • Chloroform is a possible human carcinogen and toxi. Inhalation and ingestion can be fatal. Exposure to alcohol can increase its toxic effects. Always work with this substance using appropriate gloves, lab coat and within a fume cupboard.

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