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Protein Precipitation Methods for Proteomics

 Protein Precipitation Methods for Proteomics


By Klaus D. Linse


Protein precipitation methods or protocols are used for the concentration of diluted proteins in solution. The goal when using this methods 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. Therefore protein precipitation methods are usually part of most protein analysis protocols. Protein precipitation methods can be considered as generic sample preparation methods. 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 electrostatic forces between proteins. In conventional protein precipitation protocols, this is done in vials or collection microplates.

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.
 
Note 1:         This method can also be used for larger sample volumes. To do so, one can increase the sample volume as well as the solvent volumes multi-fold by keeping the volume ratio the same.

Note 2:           (This applies to 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.

Note 3:           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. 

This method was modified after the method reported by:

Wessel and Fluegge, 1984;   A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138,141-143).

Note 4:
           If possible use only high quality chemicals.
 

Desalting/Lyophylization

  • Desalt sample by dialysis, ultrafiltration or a desalting column.
  • Freeze sample with N2 liq, Dry ice-Methanol or freezer and lyophylize sample.

This technique is just a dehydration step which does not remove contaminants.  Solvation is easier than with other methods, however, many fine precipitates often remain.  The de-salting steps are often far less effective than initially expected.


Chemicals needed              

 
I prefer to use the following brands based on our protein sequencer and mass spectrometer performance.

Acetonitrile:     EMAX0142-1, Omnisolve, EMD; HPLC, spectrophotometry, GC, Gradient Analysis, (CAS 75-05-8) EMD

Chloroform:     SIGMA (FLUKA) 25668; BioChjemika Ultra, for MB, 49:1 >99.5%, 100 ml


Isopropanol:    EMPX1838-1, Omnisolve, EMD; HPLC, spectrophotometry, GC, Gradient Analysis, (CAS 67-63-0) EMD

Methanol:        EMX0475-1 HPLC grade (CAS 67-56-1) EMD

Water:             Ultrapure water. Biosynthesis Inc.