A Collection of Protein Precipitation Methods for Proteomics

By Klaus D. Linse

Protein precipitation methods are used for the concentration of diluted proteins in solution. The goal is to purify and concentrate contaminated proteins or proteins dissolved in various matrices, buffers, detergents or from natural sources, such as blood, urine or other biofluids. The mechanism of precipitation for proteins is to alter the solvation potential of the solvent. The solubility of the solute is lowered by adding a specific reagent to manipulate the repulsive electrostaci forces between proteins.

The following is list of precipitation methods collected over several years. 

Trichloroacetic Acid (TCA) Precipitation = TCA Precipitation

• Add 1/2 volume 50% (w/v) TCA (0 ^oC) to the aqueous protein solution.

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

• Equilibrate (0 ^oC) for 10-20 min.
• Centrifuge (~5000-13,000 x g cold) to pellet protein.
• Resuspend in appropriate solution (may require neutralization).

Acetone Precipitation

·        Add acetone (-20 ^oC) at a 4:1 ratio to the aqueous protein solution.

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

·        Equilibrate (-20 ^oC) for 60 min.

·        Centrifuge (~5000-13,000 x g cold) to pellet protein.

·        Resuspend in appropriate solution.

The precipitate is often difficult to resuspend in aqueous solution.  Sonication, detergents, acetonitrile, methanol, salts, TFA (acetic or formic acid) are often used to assist in the process.  Be careful of what effects these processes have on the downstream processes.

TCA-Acetone Precipitation

·        Add 15% TCA in acetone (-20 ^oC) at a 4:1 ratio to the aqueous protein solution.

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

·        Equilibrate (-20 ^oC) for 20-60 min.

·        Centrifuge (~5000-13,000 x g cold) to pellet protein.

·        Resuspend in appropriate solution.

This technique is somewhat superior in overall recovery to TCA and Acetone separately.  The precipitate is often difficult to resuspend in aqueous solution.  Sonication, detergents, acetonitrile, methanol, salts, TFA (acetic or formic acid) are often used to assist in the process.  Be careful of what effects these processes have on the downstream processes.

Phenol, Ammonium Acetate/Methanol Precipitation

 “It’s a little tricky but I like this the best” 

            Hurkman and Tanaka, 1986, Plant Physiology 81:802-806.

• Add 1 volume of buffered (pH~8.5-9.0) phenol (e.g. 0.1 M Tris-HCl pH 8.8, 10 mM EDTA(~3 basic), 0.2% 2-mercaptoethanol/20 mM dithiothreitol, 900 mM sucrose).
• Mix for 30 min and centrifuge (~5000-13,000 x g cold) to phase separate.
• Re-extract aqueous phase (top) with 1 volume each of co-equilibrated Phenol and aqueous extraction buffers.
• Mix for 30 min, and then centrifuge (~5000-13,000 x g cold) to phase separate.
• Combine phenol phase.
• Precipitate proteins by adding 5 volumes of 0.1 M Ammonium Acetate in Methanol (-20 ^oC, 1 hr-overnight).
• Pellet protein by centrifugation (20 min 20-30 min, 14,000-20,000 x g, 4 ^oC).
• Wash pellet (2x) with 0.1 M Ammoniuim Acetate in Methanol (Resuspend 15 min, -20 ^oC; 10 min >14,000 x g, 4 ^oC).
• Wash pellet (2x) with Acetone (Resuspend 15 min, -20 ^oC; 10 min >14,000 x g, 4 ^oC).
• Wash pellet with 70 % Ethanol (Resuspend 15 min, -20 ^oC; 10 min >14,000 x g, 4 ^oC).
• Dry pellet under vacuum and store under Ar/N₂ (-20 ^oC)

This technique is superior to the other methods, particularly for plant tissues, however it is very time consuming.  The precipitate is often difficult to resuspend in aqueous solution.  Sonication, detergents, acetonitrile, methanol, salts, TFA (acetic or formic acid) are often used to assist in the process.  Be careful of what effects these processes have on the downstream processes.

Methanol Chloroform   (A)

        Wessel and Fluegge, 1984;   Anal. Biochem. 138,141-143.

·        Add 4 volumes methanol.

·        Add 3 volumes chloroform.

·        Add 3-4 volumes H₂O

·                optional: (0.1% b-mercaptoethanol or 20 mM dithiothreitol)

·        Vortex.

·        Centrifuge (~9,000 x g 10-60 min) to pellet protein between phases.

·        Discard upper phase (aqueous).

·        Add 3 volumes (original volumes) methanol

·        Centrifuge (~9,000 x g 10-20 min) to pellet protein.

·        Discard supernatant

·        Dry sample (N₂/Ar or using vacuum).

·        Resuspend in appropriate solution.

In my hands, this technique is superior to the other methods. Minimal amounts of protein (1 µg or less) are distributed over a rather large area and many proteins are, subsequently, insoluble in buffers lacking SDS. Only small quantities of chemicals or solutions are needed. Use the best grade solvents (highest purity available) for this method. 

For amino acid analysis and protein sequencing: The dried pellet can be dissolved either using pure TFA, TFA/water, heptafluoracetone (HPFA)/TFA or heptafluorpropanol (HPFA)/TFA for direct spotting on to the sample support used in the protein sequencer.

Methanol Chloroform  (B)      Modified for speed !

• In a 1.5 ml micro centrifuge tube add to 100 µl of your sample solution
  
  400 µl Methanol,
  300 µl Chloroform and
  300 to 400 µl H2O  
  

• Vortex the solution intensively and centrifuge for 10 to 20 (sometimes even 30 to 60 minutes), at about 9000 rpm (table centrifuge). Separation into two phases should be visible. The protein/peptide is found between the two phases. Discard the upper phase carefully (or keep it for further investigations or distribution studies).

• Add another 300 µl Methanol, vortex and centrifuge for 10 - 20 minutes.

• Discard the liquid and dry the precipitated pellet (with a stream of air or, better, N2 or argon, or by evaporating the remaining liquid by applying a vacuum in a speed-vac centrifuge or equivalent).

• Dissolve the sample in a buffer suitable for your next separation method and proceed.

Desalting/Lyophylization

• Desalt sample by dialysis, ultrafiltration or a desalting column.
  
• Freeze sample with N_{2 liq}, Dry ice-Methanol or freezer and lyophylize sample.