Protein Precipitation in Acetone Protocol
Reagents required in this protocol: sodium chloride, acetone
- Screw a Plug onto the base of the Filtration Cartridge.
- If your sample contains no sodium chloride, add NaCl to give a final concentration of 20 to 100 mM.
- 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 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 350 ×g (2000 rpm) ×5 minutes. Discard the flow through solvent.
- Wash the protein pellet with 400 µL acetone. Immediately centrifuge at 350 ×g (2000 rpm) ×2 minutes. Discard the flow through wash solvent.
- Proceed immediately to the Resolubilization of Intact Protein Protocol.
Resolubilization of Intact Protein Protocol
This procedure applies to samples following solvent precipitation
Reagents required in this protocol: 80% formic acid
- With Plug attached, add 150 µL of cold (-20°C) 80% formic acid in water.
- Cap the Filtration Cartridge, place in freezer for 10 minutes, then vortex or sonicate for 1 minute.
- Add 350 µL water; cap and vortex to mix the solvent.
- Intact proteins may be directly recovered in a clean Microcentrifuge Tube, centrifuging at 350 ×g (2000 rpm) ×5 minutes.
- Resolubilized proteins may also be subject to OPTIONAL SPE Protocol with the SPE Cartridge accessory (available in some trial packs).