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Protein Precipitation Procedures

  • PDF file for protein precipitation

    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
     

    Protein Precipitation Protocols

    Acetone/ TCA Precipitation
    ( works best for dilute samples)

    1 Mix 10 volumes of cold 10% TCA in acetone (stored –20C) with your samples, vortex, and incubate at –20C for at least 3hrs, but overnight is optimal.
    2 Centrifuge samples at 15000g X 10min and remove supernatant.
    3 Add the same volume of cold acetone only, vortex, and let stand at –20C for at least 10min.
    4 Centrifuge at 15000g 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 and must be removed from the sample solution prior to analysis by MS.
    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 votex 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.
    4 Spin out the precipitate at 15,000g 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 15,000g 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 2min at 15 000g – the proteins should be at the liquid interface.
    5 Remove the aqueous top layer and add 4 volumes of methanol – vortex.
    6 Centrifuge for 2min at 15 000g.
    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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    Last modified March 9, 2007 by J. Larry Campbell