Proteins run on PAGE in the absence of SDS will separate on the basis of their charge to mass ratio. While native (nondenaturing) PAGE does not provide direct measurement of molecular weight, the technique can provide useful information such as protein charge or subunit composition. Native PAGE also has the potential for separating proteins of identical molecular weight which cannot be resolved with SDS - PAGE. In addition, proteins on native PAGE usually retain their activity. This allows enzymes to be detected by sensitive and specific activity stains and delicate proteins to be resolved and recovered in a biologically active form.

The interpretation of native gels is more complex than the interpretation of SDS - PAGE gels. Not only can differences in relative mobility reflect differences in charge, mass or both, but also, proteins may have a pI at or above the pH of the buffer, in which case they will not migrate or will "retro-phorese" backward into the upper buffer chamber.

The equation governing protein mobility in native gels is:

In the presence of SDS, all proteins have the same Y_o, so that a simple relationship exists between R_f and K_R at any given T. In other words, SDS treated proteins, having identical charge to mass ratio, migrate at the same speed in free solution under electric force. With such proteins, if you know the mechanical resistance exerted by the gel, you can determine the mobility. This mechanical resistance, K_R, is directly related to molecular weight, so that a determination of K_R allows calculation of molecular weight. In native gels, the situation is more complicated. Both Y₀ and K_R can vary between proteins. Y₀ is related to the charge, while K_R varies with the mass.

Separation of protein mixtures and protein standards on gels of varying percentages allows the determination of both charge and mass of the sample proteins. The graphic analysis used is known as the Ferguson Plot. On the Ferguson Plot, log R_f is graphed vs %T for a range of %T (the originators of this system covered 7 different % gels). By the above equation, the graph should have a slope of K_R and a Y intercept (at %T = 0) of Y_o. Comparison with standards of known charge and size allows determination of the charge and molecular weight of the samples.

Another less laborious way of simplifying the interpretation of Native PAGE gels is to run a gradient gel. Native gradient gels are poured in the same manner as gradient SDS PAGE gels. As proteins migrate through the increasing acrylamide concentration, into regions of ever smaller pore sizes, their mobility decreases. Eventually, each protein reaches its "pore-limit", at which point it slows to a minimum migration rate, which is constant for all proteins at their pore limit. The band pattern is stabilized at this point, so that gradient Native gel PAGE approaches an equilibrium system, in that beyond a certain run length, only minimal changes occur in the gel pattern.

Once proteins reach their pore limit, their relative positions are a direct reflection of their molecular weight. In a linear gradient, log MW is proportional to log R_f over a wide range, although the curve is actually sigmoid in shape. This type of analysis is more subject to artifacts than the Ferguson Plot, but is easier to carry out.

Finally, native gradient gels may be analyzed with activity stains, which simplify the pattern by only visualizing enzyme activities of interest. This can be useful when purifying an enzyme from a mixture of isozymes, or when studying the expression or activity of enzyme families. It also allows for the isolation of electrophoretically pure, active enzyme. Used in conjunction with gradient gels, activity stains can provide molecular weight information about otherwise uncharacterized enzymes. Of course, the technique is limited to enzymes for which chromogenic or chemiluminescent assays exist. Often an assay can be adapted from existing techniques with little effort. The critical requirement is that a colored compound be deposited in the gel in an insoluble form, either by the enzyme (positive stain) or in a reaction inhibited by the enzyme (negative stain).

NEXT TOPIC: Sample Preparation for Native Protein Electrophoresis