Top-Down Workflow Protocols

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.

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).