Acetone precipitation is a technique for isolating and purifying biomolecules. Its primary function is to concentrate proteins or nucleic acids from a solution and separate them from interfering substances like salts and detergents. By adding acetone, researchers cause these larger molecules to become insoluble and precipitate, allowing for their collection.
How Acetone Precipitation Works
The effectiveness of acetone precipitation lies in its ability to alter the chemical environment surrounding biomolecules. In a water-based solution, proteins are kept dissolved because water molecules form structured layers, known as hydration shells, around them. Acetone, an organic solvent, is less polar than water and disrupts these hydration shells, reducing the protein’s solubility.
As the acetone concentration increases, the solvent becomes less capable of keeping the protein molecules dispersed. This encourages the proteins to interact with each other, leading them to aggregate and form a solid precipitate. Performing this process at cold temperatures, like -20°C, further decreases protein solubility and protects their structural integrity.
Standard Acetone Precipitation Steps
The procedure begins with preparing the initial sample and cooling the necessary volume of acetone to -20°C. The cold acetone is then added to the protein sample in an acetone-compatible tube. A standard ratio is four volumes of cold acetone for every one volume of the protein solution. The mixture is then thoroughly vortexed.
Following mixing, the sample is incubated at -20°C for a period from 60 minutes to overnight, depending on the protein concentration. This allows the proteins to precipitate fully. The mixture is then centrifuged at high speeds, between 13,000 and 15,000 x g, for 10 to 15 minutes to pack the proteins into a dense pellet.
Once centrifugation is complete, the liquid supernatant, containing unwanted contaminants, is carefully decanted without disturbing the protein pellet. For increased purity, an optional washing step can be performed by adding cold 80-100% acetone and recentrifuging. The pellet is then air-dried for 5-10 minutes to evaporate residual acetone before being redissolved.
Optimizing Your Acetone Precipitation
Adjusting several variables can improve the outcome of acetone precipitation. The ratio of acetone to the sample is a primary factor; increasing the acetone percentage beyond the standard 4:1 ratio can enhance recovery. The initial protein concentration also matters, as dilute samples may require longer incubation times or a carrier to facilitate pellet formation.
Temperature and incubation time are also adjustable. While -20°C is standard, precipitation can occur at room temperature if the salt concentration is optimized. Adding a small amount of salt, like sodium chloride to a final concentration of 1-30 mM, can increase protein recovery. The purity of the acetone is another consideration.
If salts or detergents are co-precipitating with the protein, adjusting the acetone concentration or performing additional wash steps can help remove these contaminants. For very small pellets, adding a carrier protein or dye can aid in visualization, though this must be compatible with any subsequent analysis.
Solving Common Precipitation Problems
A frequent issue is low or no visible pellet, indicating poor protein recovery. This can happen if the initial protein concentration is too low or the incubation time is too short. Extending the incubation period, increasing the acetone-to-sample ratio, or adding a small amount of salt can help improve the yield.
Another common problem is difficulty re-solubilizing the dried pellet. Over-drying the pellet can make it nearly impossible to dissolve, so air-dry for just enough time to remove the acetone smell, typically not more than 10 minutes. If the pellet will not dissolve, using a stronger buffer with detergents or chaotropic agents can aid in resuspension.
Contamination of the final product with salts or other small molecules can also occur. This is common when the initial sample has a high concentration of these substances. Performing one or two additional wash steps with cold acetone is an effective way to remove these contaminants.
Next Steps After Precipitation
Once the protein pellet is precipitated and dried, the next step is resuspension. The choice of buffer for this is dependent on the downstream application. For example, samples for SDS-PAGE are often resuspended in a Laemmli buffer, while those for mass spectrometry may require a buffer containing urea. Gentle techniques like pipetting or light vortexing are usually sufficient, but stubborn pellets may require brief sonication.
It is often useful to assess the recovery and purity of the precipitated protein. Running a small portion of the resuspended sample on an SDS-PAGE gel can provide a quick visual confirmation of the protein’s presence and an estimate of its purity. Comparing the precipitated sample to the starting material can also give an indication of yield.
The resuspended sample must be compatible with subsequent analytical methods. Residual acetone must be completely removed as it can interfere with certain assays. The resuspension buffer should not inhibit enzymes or interfere with immunoassays. For storage, proteins can be kept at 4°C for short-term use or frozen.