Hydrophobic Electrophoresis

Although aqueous buffers are the appropriate solvents for gel electrophoresis of native hydrophilic macromolecules, separations of hydrophobic species require the presence of detergents or miscible organic solvents. Uncharged or amphoteric detergents are compatible with the formation of a gel and electrophoresis. Alternatively, charged detergents can be added to the buffer and have a negligible effect on electrophoresis, so long as conductance is not greatly elevated. The most widely used detergent for that purpose is the strongly dissociating and denaturing detergent, SDS. However, the molecular sieving of charged detergent micelles in electrophoresis as a function of gel concentration needs to be considered, as well as the generation of moving boundaries in discontinuous buffer systems, with the detergent as a leading or trailing ion. Miscible organic solvents are particularly suitable for relatively hydrophobic gels, such as some of the “Hydrolink” gels (1) or the new spongelike copolymer gel media(2).

When it is desirable to preserve native or relatively undissociated structures and their biological activities, gel electrophoresis must be conducted in “nondenaturing detergents,” ie, under conditions that render the particular detergent relatively innocuous with regard to the disruption and inactivation of active structures and complexes. For that purpose, seven groups of amphoteric or uncharged detergents are particularly useful(3). Their effectiveness in solubilization depends sharply on their concentration, critical micelle concentration (cmc) and micellar weights, and the protein/detergent ratio.

References

1. R. J. Molinari, M. Connors, and R. G. L. Shorr (1993) Adv. Electrophoresis 6, 44–64. 

2. M. G. Harrington, K. H. Lee, E. Bailey, and L. E. Hood (1994) Electrophoresis 15, 187–199. 

3. L. M. Hjelmeland and A. Chrambach (1984) Methods Enzymol. 104, 305–318.