The Ins and Outs of Protein Concentration – Protein Precipitation

While precipitation is an obvious choice for concentrating DNA and RNA samples, it can also be an effective way to concentrate proteins. Here in installment two of this three part series, I describe the two most common methods for protein precipitation – ammonium sulfate and trichloroacetic acid.


Precipitation of proteins occurs primarily by hydrophobic aggregation, either by subtly disrupting the folded structure of the protein and exposing more of the hydrophobic interior to the solution, or by dehydrating the shells of water molecules that form over hydrophobic patches on the surface of properly folded proteins. Once the proteins start aggregating into larger structures, the amount of water per protein drops, enhancing the density differences between the proteins and the solute. Once these aggregates grow large enough, and if there are enough of them, they disrupt the path of light through the solution, giving it a cloudy appearance. In addition, the density differences become great enough for the aggregates to be readily pelleted in the centrifuge. Because these methods depend on the protein molecules “finding” one another in solution and forming these aggregates, the efficiency of this method depends on the concentration of the protein being precipitated – the lower the concentration, the harder it is to form aggregates. There are two practical reagents used to precipitate proteins: ammonium sulfate and trichloroacetic acid.

Ammonium sulfate

How it works.  Salting proteins out of solution was actually discovered over 120 years ago by Franz Hofmeister when he noticed that the addition of different salts caused precipitate to form in solutions of egg whites. He ordered the anions and cations by their ability to precipitate proteins in what is known as the Hofmeister series. Biochemistry has spent the 120 years since using this trick to purify proteins while also arguing about how it works. The current, mostly-accepted mechanism says that salting proteins out of solution occurs when the water molecules are titrated away from the solvent shells around the protein to the solvent shells around the ions that make up the salt. Salts high in the Hofmeister series are the most efficient at protein precipitation because of the large, stable solvent shells they maintain. This increases the surface tension of the solution, which effectively increases the hydrophobic effect, which stabilizes the protein structure while also encouraging the hydrophobic regions on the surfaces of different molecules to interact, affecting aggregation. Because of this, proteins with a larger amount of hydrophobic surface character precipitate at lower salt concentrations than one with little hydrophobic surface character, and these protein to protein differences are exploited during protein purification procedures.

In practical terms.  The go-to salt for purification or protein concentration is ammonium sulfate, since both the anion and cation are both high in the Hofmeister series. This salt has a high solubility at around 4M, with most proteins precipitating by the time the salt concentration reaches 3.2M. These concentrations can be obtained either by adding the salt directly to the protein solution or by the addition of a calculated volume of a saturated solution of the salt. Adding the salt directly to the protein solution helps keep the total volume of the solution under control, but can be problematic if the volume of the initial sample is low. Once the salt is dissolved or the solutions are mixed, the protein is allowed to precipitate for 30-60 minutes on ice (longer if the protein concentration is low) before pelleting the protein in a centrifuge. Because salting out stabilizes the protein structure, the pelleted protein will almost always readily re-dissolve into a buffer (lacking ammonium sulfate) with enzymes maintaining their specific activity (some measure of activity/mass of protein). However, even though salting out occurs via a phase transition mechanism, some quantity of salt will come down with the protein, leaving you with an undefined solution once you re-dissolve the protein. This requires dialysis or a de-salting (size exclusion) column to move the protein into a defined solution, which could result in some dilution of your now-concentrated protein.

Trichloroacetic acid

How it works. Proteins can be efficiently precipitated with trichloroacetic acid (TCA), acetone, or even ethanol, although the concentrations at which these (mostly) miscible organic solvents function can vary greatly. As with ammonium sulfate, the mechanism of precipitation is hydrophobic aggregation. However, in addition to disruption of the solvation layers of the proteins, these compounds also partially denature the proteins, exposing even more hydrophobic surface to the solvent.

In practical terms.  TCA is often the compound of choice amongst the miscible organics because it is effective at lower concentrations than the others – ~15% for TCA, ~75% for acetone, and ~90% for ethanol – which means the sample volume doesn’t increase dramatically. Because of this, the protein concentration remains higher during a TCA precipitation, increasing the efficiency of the precipitation. However, since TCA (as well as the others) partially denature the protein, there is a good chance that your protein of interest won’t dissolve back into an aqueous buffer in the absence of a detergent (SDS); and even if it does, it may have a markedly lower specific activity. For this reason, these methods are normally only employed with samples that don’t require functional enzymes, such as SDS-PAGE or mass spectrometry analysis. Because TCA is an acid, the protein pellet is either washed with 75% acetone to remove the TCA, or base is added after the pellet is resuspended in SDS-PAGE sample buffer (until the bromophenol blue turns from yellow back to blue) to neutralize the pH. However, if you happen to be lucky and have a protein that does tolerate this treatment, this method has the advantage of precipitating the protein with a minimum of salt in the pellet, possibly eliminating follow-up desalting procedures, depending on your application.

As always with proteins, your results may vary; but for many proteins precipitation has proven to be a good concentration method. As always, if you have any tips, hints, or suggestions to share, please speak up in the comments section!  Next up: protein concentration via chromatography.

This is part 1 of a 3 part series on the in’s and out’s of protein concentration:

Part 1: Semi-permeable membranes

Part 2: Protein precipitation

Part 3: Chromatography


  1. Marc on June 8, 2016 at 1:24 am

    Actually, ‘effecting’ is correct, if you meant it in the sense ‘causing x to happen’. ‘Encouraging’ might be a better word. ‘Affecting aggregation’ is less informative, since you mean to say that the hydrophobic surface, in combination with the more ordered water environment, favors aggregation.

    I’m a word nerd.

  2. Dave on December 7, 2015 at 10:40 am

    Great article thanks a lot for posting.

    I noticed something:
    “hydrophobic regions on the surfaces of different molecules to interact, effecting aggregation”
    – Should be affecting!

    • Dr Amanda Welch on December 7, 2015 at 10:55 am

      Great catch! Thanks!

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