Team:Cambridge/Protocols/Acetone Precipitation of Proteins

Acetone precipitation of proteins

After protein purification it is necessary to remove chemicals retained from the elution buffers and to concentrate the protein sample to allow downstream processing.

Theory

Proteins are insoluble in acetone (particularly at low temperatures) whilst many small molecules which could interfere with downstream protein work are soluble. By precipitating proteins in this solvent you can remove buffer contaminant and concentrate protein into a pellet which can be redissolved by other solvents.

Practice

1. Approx 30 min before you start, cool a sample of acetone 4 times the volume of the protein solution you wish to precipitate in a -20 freezer.
2. Spilt protein sample into centrifuge tubes for the benchtop centrifuge.
3. Add cold acetone to the protein sample.
4. Vortex tubes to mix.
5. Incubate for 60 minutes at -20.
6. Centrifuge for 10 minutes at 13 000 xG
7. Decant supernatant - try not to disturb the pellet.
8. Leave the lids off the tubes to let remaining acetone evaporate.
9. Redissolve in whatever buffer the downstream processes require.

Safety

Acetone is flammable - decant only small volumes, keep stock bottle in flammable substances cupboard. Inhalation can cause irritation. Wear gloves and labcoats, keep away from sources of ignition. Do not mix with peroxide containing substances.