Methanol/Chloroform Precipitation Method
1. Add equal volume (100 uL) of 100% methanol and 62.5 uL of chloroform to 100 uL of soluble Protein and mix well for 5 min.
2. Centrifuge at 12,000_ g for 15 min.
3. Remove supernatant without attaching the interface layer (protein fraction) by pipette.
4. Add 100 uL of 100% methanol to the sample and mix gently for 5 min. Centrifuged at 12,000_ g at 25 _C for 15 min.
5. The supernatant is discarded, then the pellet air-dried.
Acetone Precipitation Method
1. Cool the required volume of acetone to -20°C.
2. Place protein sample in acetone-compatible tube.
3. Add four times the sample volume of cold (-20°C) acetone to the tube.
4. Vortex tube and incubate for 4 hours at -20°C (can be kept overnight).
5. Centrifuge 10 minutes at 13,000-15,000 × g.
6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
Optional: If additional cycles of precipitation are necessary to completely remove the interfering substance, then repeat
steps 2-5 before proceeding to step 7.
7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it
may not dissolve properly.
8. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protein pellet.