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 Protein Digestion Protocol.
Protein Digestion Protocol
Reagents required in this protocol: 8M Urea, 100 m M Tris pH 8.0, Reducing agent (e.g. 200 mM DTT or TCEP or alternate), alkylating reagent (e.g. 200 mM iodoacetamide or alternate), Trypsin, 10% trifluoroacetic acid (TFA)
- With Plug attached, add 100 µL 8 M urea to a precipitated sample.
- Cap the Filtration Cartridge, vortex 30 seconds, and then sonicate an additional 10 minutes. Let sit at room temperature for an additional 30 minutes.
- Add 400 µL of 100 mM Tris buffer (pH 8), cap and vortex briefly to mix.
- Optional: Reduce/alkylate disulfide bonds as per standard protocols (e.g. dithiothreitol (DTT)/iodoacetamide).
- Add tryspin at 50:1 (protein: enzyme by mass). Cap the device, incubate in a warm water bath as per conventional solution digestion (e.g. 37°C, 1 hour to overnight).
- Stop the reaction by acidifying with trifluoroacetic acid (TFA) (final 1%).
- Peptides may be recovered by centrifuging (Plug removed, 2500 ×g (5000 rpm) ×5 minutes), or subject to OPTIONAL SPE cleanup with the SPE Cartridge accessory (available in some trial packs).