Protein Precipitation Procedures

Notes:

• It should first be noted here that the best possible outcome for a protein sample
involves no protein precipitation at all as there is always some degree of loss. If
possible, avoid doing any of the following protocols unless dilution of the sample,
dialysis, or buffer exchange into a compatible buffer system cannot be achieved.
• The solutes which you are trying to remove by precipitation must be soluble in the
reagents corresponding to the procedure used.
• All volumes based on original sample volume.

Chemical reagents (Reagents should be HPLC or USP grade):

• Trichloroacetic acid (TCA)

• Deoxycholate (DOC)
• Acetone
• Methanol
• Chloroform

Precipitation protocols:

• Acetone/ TCA Precipitation

• TCA / DOC Precipitation
• Chloroform / Methanol Precipitation

Acetone/ TCA Precipitation – works best for dilute samples

1. Mix 10 volumes of cold 10% TCA in Acetone (stored –20oC) with your samples,
vortex, and incubate at –20oC for at least 3hrs, but overnight is optimal.
2. Centrifuge samples at 15000xg X 10min and remove supernatant.
3. Add the same volume of cold Acetone only, vortex, and let stand at –20oC for at
least 10min.
4. Centrifuge at 15000xg X 5min, remove supernatant and allow pellets to air dry.
Care should be taken to prevent complete desiccation of the protein pellet as this
will make re-solubilization much harder.

TCA / DOC Precipitation

Notes: DOC is a detergent – no good for direct use on MS. It is best for before 1 and 2D
gels analysis.
1. Mix your sample with 1/100 of its volume of 2% DOC and incubate on ice for 30
minutes.
2. Add enough 100% TCA to bring your sample to a final TCA concentration of 15%
and vortex immediately for 30 seconds to prevent any large conglomerates from
forming that may trap the contaminants you are trying to remove.
3. Let the sample precipitate for a minimum of one hour, but preferably overnight.

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 4. Spin out the precipitate at 15,000xg for 10 minutes and aspirate the TCA and
soluble contaminants from the pellet.
5. Wash the pellet with ice cold ethanol or acetone making sure to break up the
pellet to the point that it is getting washed.
6. Vortex and let sit at room temp for 5 minutes.
7. Spin down the pellet at 15,000xg for 10 minutes and aspirate the supernatant off
of the pellet.
8. Repeat the wash step and re-spin down the pellet.
9. Dry the pellet under a SLOW stream of nitrogen and make sure not to bring the
pellet to complete dryness as it will be difficult to bring back into solution.

Chloroform / Methanol Precipitation

1. Add 4 volumes of methanol and vortex well.

2. Add 1 volume of chloroform and vortex.
3. Add 3 volumes of dH2O and vortex
4. Centrifuge for 2 min at 15 000xg – the proteins should be at the liquid interface
5. Remove the aqueous top layer and add 4 volumes of methanol – vortex.
6. Centrifuge for 2 min at 15 000xg
7. Remove as much liquid as possible without disturbing precipitate.
8. Speed-Vac sample to dryness or dry under nitrogen.

Protein Re-solubilization

The protein precipitates need to be re-solubilized in a buffer compatible with the next
step of analysis. For example: SDS-PAGE sample buffer for 1D gel analysis, re-hydration
buffer for 2D-gel analysis, or a small volume of fresh buffered 6M urea then diluted to an
appropriate concentration for an in-solution digests and protein quantification.

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