Download Protein Precipitation Protocol (PDF)

Protein Precipitation in Acetone Protocol

PREPARATION NOTES
The capacity of the ProTrap XG Filtration Cartridge is 250 µg of protein.
If utilizing the SPE Cartridge (see SPE Protein/Peptide Clean-Up Protocol) the maximum amount of protein/peptides to load on the SPE Cartridge is 50 µg.
Spin speeds are based on a standard benchtop microcentrifuge with 24 x 1.5/2.0 mL rotor.
Times provided are guidelines only.
If more than a few microliters of liquid remains in the Filtration Cartridge after any spin, return it to the centrifuge and repeat the spin, or consider increasing the spin speed. 3000 ×g (6000 rpm) is recommended for subsequent spins and the ProTrap XG has been tested up to 9000 ×g (10,000 rpm).

MATERIALS REQUIRED
All chemicals and reagents should be ACS grade/HPLC grade or better.
Acetone
5 M NaCl in water

PROTEIN PRECIPITATION

  • To your protein solution, before adding it to the Filtration Cartridge, add sufficient 5 M NaCl to bring the NaCl concentration to 50 mM before adding your sample to the Filtration Cartridge.
  • Screw a Plug onto the base of the Filtration Cartridge.
  • Transfer 100 µL of the salted protein to the plugged Filtration Cartridge.
  • Add 400 µL room temperature acetone.
  • Cap the Filtration Cartridge and rock gently, tilting no more than 45°, to combine the solvents. 
  • Insert the Filtration Cartridge in the Microcentrifuge Tube, allow 30 minutes for the protein to fully aggregate at room temperature.
  • With Plug attached, centrifuge at 2500 ×g (5000 rpm) ×2 minutes.
  • Remove Filtration Cartridge from the Microcentrifuge Tube, invert, and unscrew the Plug.
  • Return the capped Filtration Cartridge to the Microcentrifuge Tube and centrifuge at 500 ×g (2000 rpm) ×5 minutes.  Discard the flow through solvent.  If any solvent remains in the Filtration Cartridge, re-spin the unit for 2-5 minutes at 3000 ×g (6000 rpm).
  • Wash the protein pellet with 400 µL acetone.  Immediately centrifuge at 500 ×g (2000 rpm) ×2 minutes. Discard the flow through wash solvent.

Intact proteins may be resolubilized using the Resolubilization of Intact Protein Protocol , or subject to enzymatic digestion within the Protein Digestion Protocol.

Do not allow the membrane in the Filtration Cartridge to dry out between steps.  Proceed immediately to your next protocol.

Download Protein Precipitation Protocol (PDF)

GO TO Resolubilization of Intact Protein Protocol

GO TO Protein Digestion Protocol