Protein Precipitation in Acetone Protocol
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).
All chemicals and reagents should be ACS grade/HPLC grade or better.
5 M NaCl in water
- 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.
Do not allow the membrane in the Filtration Cartridge to dry out between steps. Proceed immediately to your next protocol.