APL has offered native gel analysis services since 1998. Today many labs unfortunately ignore this valuable tool because they think native gels are just too hard to use, or because they mistakenly believe they can only be used with acidic proteins. At Alliance Protein Laboratories we routinely run native gels on both basic and acidic proteins. We would be happy to run samples for you and/or work with you to develop a protocol for use in your lab.
What is a native gel?
"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.
The electric charge driving the electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. This charge will, of course, depend on the amino acid composition of the protein as well as post-translational modifications such as addition of sialic acids.
Since the protein retains its folded conformation, its hydrodynamic size and mobility on the gel will also vary with the nature of this conformation (higher mobility for more compact conformations, lower for larger structures like oligomers). If native PAGE is carried out near neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation, self-association or aggregation, and the binding of other proteins or compounds.
Thus native gels can be sensitive to any process that alters either the charge or the conformation of a protein. This makes them excellent tools for detecting things such as:
changes in charge due to chemical degradation (e.g. deamidation)
unfolded, "molten globule", or other modified conformations
oligomers and aggregates (both covalent and non-covalent)
binding events (protein-protein or protein-ligand)
These properties, and their relatively high throughput, make native gels excellent tools for analyzing accelerated stability samples, demonstrating comparability of different lots or processes, or examining the effects of excipients.
Another advantage of native gels is that it is possible to recover proteins in their native state after the separation. Recovery of active biological materials may, however, need to be done prior to any fixing or staining.
Note that the native gels being discussed here are now sometimes called "clear native" gels. They differ in principle from so-called "blue native" gels, which depend on the binding of dye to give the protein a very high electric charge, overwhelming the intrinsic charge on the polypeptide. If the amount of dye bound is assumed to be proportional to the mass of the protein then the mobility observed using this "blue native" protocol can be used to estimate the molar mass by comparing to protein standards, much as is done for SDS-PAGE. The mass standards sold for use with blue native gels cannot be used to estimate molar masses for the true native gels being discussed here.
Example Applications for Native Gels
Below are several examples of native gel applications.
For acidic proteins, Laemmli's gel system without SDS can be used for native PAGE. An example is shown above. The glycoprotein used here, recombinant human erythropoietin (EPO, lane 1) is highly sialylated and hence negatively charged at the pH of Laemmli's system, pH=8.4. This gel also shows the results from native PAGE for EPO samples after being stressed by heating at 79 °C. As the incubation time is increased (lanes 2-5) there is increasing formation of a new band, corresponding to dimers, as confirmed by both sedimentation velocity and non-reducing SDS PAGE analysis.
It is therefore possible to screen conditions that minimize such oligomer formation using native PAGE. In fact, the native gel above shows no dimer formation in histidine, glycine or Tris-HCl buffers (all at 20 mM), consistent with the highly-reversible thermal unfolding of EPO in those conditions.
The gel shown above is another example of native PAGE for an acidic protein, BSA, obtained from 2 different sources. In this case, the gel was run at 60 °C to examine the actual events occurring during unfolding of BSA at 60 °C. Many bands are observed for BSA after heating in buffer alone (lanes 1 and 4, from different suppliers). Fewer bands are observed when the BSA sample contains detergents (i.e., it is protected by the detergents), as in lanes 2-3 and 5-7.
For basic proteins, acid-urea gels or acid gels are often used. However, most proteins denature to some extent at acidic pH and in the presence of urea. Under such denaturing conditions, the mobility of proteins may not reflect their conformation at physiological pH. In addition, protein-protein interactions or aggregation of proteins that occurs at normal pH may be altered. Therefore, we run native gels for basic proteins under more physiological conditions, i.e., pH 6.1 and in the absence of urea.
The native gel above shows an example with a protein that has an isoelectric point (pI) above 8. Lanes 1-6 correspond to the same protein processed or stored differently. Only one sample shows an extra band with higher mobility, reflecting some form of chemical degradation under this particular condition. Therefore, one can use native-PAGE to screen conditions that eliminate such degradation. Since the mobility of the degradation product is greater, it should have more positive charges than the starting protein at the pH of the gel, pH=6.1.
This native PAGE system for basic proteins can detect aggregation as with Laemmli's system. As an example, the same protein shown in the previous gel was heated in 5 different buffers and examined for the degree of aggregation. As shown by the native gel above, the band corresponding to the monomer (highest mobility) decreases and new bands corresponding to aggregates appear upon heating, while only one band is observed in these buffers before heating (as in lanes 1-3 in Fig. 4). The degree of aggregation is highly dependent on the type of buffer. In one condition (lane 5), almost no aggregation occurred, indicating that under this condition aggregation of this protein is greatly reduced.
Some of our native gel publications and presentations
"Aggregation analysis of therapeutic proteins, part 1: General aspects and techniques for assessment". Arakawa, T., Philo, J. S., Ejima, D., Tsumoto, K., and Arisaka, F. (2006). Bioprocess International 4 (10), 42-49
"Kinetic and Thermodynamic Analysis of Thermal Unfolding of Recombinant Erythropoietin". T. Arakawa, J. S. Philo and Y. Kita (2001). Biosci. Biotechnol. Biochem. 65, 121-1327
"Effects of Additives on Reversibility of Thermal Unfolding " [poster, WCBP meeting, 2000]
"Binding of Neu differentiation factor with the extracellular domain of Her2 and Her3". Horan, T. P., Wen, J., Arakawa, T., Liu, N., Brankow, D., Hu, S., Ratzkin, B., and Philo, J. S. (1995). J. Biol. Chem. 270, 24604-24608.
Arakawa, T. and Kita, Y. (2000). Protection of bovine serum albumin from aggregation by Tween 80. J. Pharm. Sci. 89, 646-651
Kita, Y. and Arakawa, T. (2002). Salts and glycine increase reversibility and decrease aggregation during thermal unfolding of ribonuclease-A. Biosci. Biotechnol. Biochem. 66, 880-882.
Wen, J., Zhang, M., Horan, T. P., Philo, J. S., Li, T. S., Wypych, J., Mendiaz, E. A., Langley, K. E., Aoki, K. H., Kuwamoto, M., Kita, Y., and Arakawa, T. (2001). Copper staining method for extracting biologically active proteins from native gels. Biosci. Biotechnol. Biochem. 65, 1315-1320.