Precipitation Procedures

Protein precipitation is an optional step in sample preparation for 2-D electrophoresis. Precipitation, followed by resuspension in sample solution, is generally employed to selectively separate proteins in the sample from contaminating species such as salts, detergents, nucleic acids, lipids, etc., that would otherwise interfere with the 2-D result. Precipitation followed by resuspension can also be employed to prepare a concentrated protein sample from a dilute source (e.g. plant tissues, urine). Note, however, that no precipitation technique is completely efficient, and some proteins may not readily resuspend following precipitation. Thus, employing a precipitation step during sample preparation can alter the protein profile of a sample. When complete and accurate representation of all the proteins in a sample is of paramount interest, precipitation and resuspension should be avoided.

2-D Clean-Up Kit from Cytiva can be used to remove contaminating substances and improve the 2-D electrophoresis pattern. Proteins are precipitated with a combination of precipitation reagents while the interfering substances, such as nucleic acids, salts, lipids, or detergents, remain in solution. Samples can be resuspended in the desired denaturing solution for IEF. Each kit can process 50 samples of up to 100 µL each. Section 1.4.1 (Cleaning up samples using 2-D Clean-Up Kit) describes the kit and provides a protocol for use.

Table 8 lists some of the precipitation techniques that can be used. If sample preparation requires precipitation, typically only one precipitation technique is employed.

Table 8. Precipitation procedures.

Precipitation method	General procedure	Limitations
Ammonium sulfate precipitation (“Salting out”) In the presence of high salt concentrations, proteins tend to aggregate and precipitate out of solution. Many potential contaminants (e.g. nucleic acids) will remain in solution.	Prepare protein so that the final concentration of the protein solution is > 1 mg/mL in a buffer solution that is > 50 mM and contains EDTA. Slowly add ammonium sulfate to the desired percent saturation (44) and stir for 10–30 min. Pellet proteins by centrifugation.	Many proteins remain soluble at high salt concentrations, so this method is not recommended when total protein representation is desired. This method can, however, be used for prefractionation or enrichment. Residual ammonium sulfate will interfere with IEF and must be removed (45). See section 1.5 on removal of salts (Desalting samples using Mini Dialysis Kit).
TCA precipitation TCA (trichloroacetic acid) is a very effective protein precipitant.	TCA is added to the extract to a final concentration of 10–20% and the proteins are allowed to precipitate on ice for 30 min (46). Alternatively, tissue may be homogenized directly into 10–20% TCA (35, 47). This approach limits proteolysis and other protein modifications. Centrifuge and wash pellet with acetone or ethanol to remove residual TCA.	Proteins may be difficult to resolubilize and may not resolubilize completely. Residual TCA must be removed by extensive washing with acetone or ethanol. Extended exposure to this low pH solution may cause some protein degradation or modification.
Acetone precipitation This organic solvent is commonly used to precipitate proteins. Many organic-soluble contaminants (e.g. detergents, lipids) will remain in solution.	Add at least three volumes of ice-cold acetone to the extract. Allow proteins to precipitate at -20 ºC for at least 2 h. Pellet proteins by centrifugation (46, 48–50). Residual acetone is removed by air-drying or lyophilization.	Incomplete recovery of all proteins. Compatibility of acetone with tubes may be an issue.
Precipitation with TCA in acetone The combination of TCA and acetone is commonly used to precipitate proteins during sample preparation for 2-D electrophoresis, and is more effective than either TCA or acetone alone.	Suspend lysed or disrupted sample in 10% TCA in acetone with either 0.07% 2-mercaptoethanol or 20 mM DTT. Precipitate proteins for at least 45 min at -20 °C. Pellet proteins by centrifugation and wash pellet with cold acetone containing either 0.07% 2-mercaptoethanol or 20 mM DTT. Remove residual acetone by air drying or lyophilization (5, 28, 34, 43, 51, 52).	Proteins may be difficult to resolubilize and may not resolubilize completely. Extended exposure to this low pH solution may cause some protein degradation or modification.
Precipitation with ammonium acetate in methanol following phenol extraction This technique has proven useful with plant samples containing high levels of interfering substances.	Proteins in the sample are extracted into water- or buffer-saturated phenol. Proteins are precipitated from the phenol phase with 0.1 M ammonium acetate in methanol. The pellet is washed several times with ammonium acetate in methanol and then with acetone. Residual acetone is evaporated (42, 43, 47, 53).	The method is complicated and time

For an overview of precipitation techniques, see references 17, 18, and 44.