Nondenaturing Gel Electrophoresis Of Proteins

This protocol was contributed by Jessica Habashi and Xing-Wang Deng (Yale University).

Nondenaturing (or native) electrophoresis is used for analyzing proteins in their native conformations. Since there is no SDS or heat treatment, the protein loaded onto the native gel is in a form most similar to that in the cell (i.e., folded, in a complex with itself or other partners). Thus, although the level of resolution of the bands is not always as good as in conventional denaturing SDS-PAGE, the native gel can be a useful tool for characterizing proteins and their complexes. For background on nondenaturing gel electrophoresis, see Gallagher (1995).

The most important technical aspect of native gel electrophoresis is the choice of the appropriate pH for the gel solutions. Since there is no SDS in this procedure to confer a net negative charge to the protein of interest, the pH must be relied upon for conferring a net negative charge. If one considers the amino and carboxyl groups that populate the side chains of the amino acids, a higher pH is more likely expected to give a protein a net negative charge. Excessively basic conditions should be avoided, however, as these will cause the protein to denature and even precipitate.

For animal proteins, a pH range between 6.0 and 9.0 is usually used as a starting point, because this will be closest to physiological conditions. For plants, it may be necessary to estimate the isoelectric point (pi) of the protein using protein analysis software and to choose a pH range accordingly. Once determined, a pH of the gel solutions above the pi will confer a net negative charge on the protein. Please note that in the protocol below, the desired pH is adjusted to -8.0.

Preparing Protein Extracts from Plant Seedlings

The protein extraction procedure is the same as that for regular SDS-PAGE, but the buffers lack EDTA, SDS, and (3-mercaptoethanol, and the samples are not boiled prior to loading. Please note that depending on the nature of the protein, the initial preparation may need to be done under altered conditions. This protocol was developed for a light-labile protein complex, which requires the use of a green safelight.

Materials

Grinding buffer

10% glycerol 400 mM sucrose 50 mM Tris (pH 8.0)

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