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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).

Download Top-Down Workflow Protocols (PDF)