PROTOCOL

ACETONE PRECIPITATION OF PROTEINS

This protocol can be used to precipitate proteins from solutions where heavy detergents were used to prepare protein extracts.

Required materials:

  • Cold (-20°C) acetone (volume = 4X that of the protein samples to be precipitated).
  • Centrifuge tubes made of acetone-compatible polypropylene and able to hold 5X sample volume.
  • Centrifuge and rotor for the tubes used (min speed 18000 x g).
  • 1M Urea/50 mM NH4HCO3.

Procedure:

  1. Cool required volume of acetone to -20°C.
  2. Place protein sample into acetone-compatible tube.
  3. Add 4 fold volume of cold acetone to protein mixture.
  4. Vortex and incubate at -80°C for 1 hour (or overnight)
  5. Centrifuge @ 18,000g for 10 minutes
  6. Decant and properly dispose of supernatant, keeping in mind to not disturb the protein pellet.
  7. Allow pellet to air dry for 10-30 minutes in uncapped tube. Do not over-dry the pellet as it may not re-dissolve properly.
  8. Re-suspend the pellet in 50-100μL 1M Urea/50mM NH4HCO3.
  9. Bring sample to SPARC facility OR, proceed with digestion.

 

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