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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.
Materials
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