Isoelectric Focusing of Biological Particles

Gregory G. Wolken and Dr. Edgar A. Arriaga, University of Minnesota, Minneapolis, MN

A PDF version is available in the Fall 2011 AES Newsletter.

What electrophoretic separation method can be used to separate different species of bacteria, fractionate subcellular components of a cell homogenate, test the purification of a virus from an environmental sample, and more? Isoelectric focusing (IEF) is a powerful technique for the analysis of biological particles such as cells, organelles, and viruses.1 This flexible technique can be used for separation and fractionation, functional measurements, and discernment of the chemical properties of biological particles.

Isoelectric focusing is a technique that separates analytes based on their isoelectric point (pI), the pH at which they have a net neutral charge. Species migrate through a pH gradient to their pI, Gaylord Hotel where they are focused. The pI of a small molecule, protein, or particle depends on the chemical composition of its ionizable functional groups (Fig. 1). Therefore, IEF-based techniques are not only useful as separation methods, but can provide valuable information about the chemical features of the species being separated.

Figure 1. Origin of pI of a biological particle. Biological parti- cles such as cells (1) have membranes (2) made of phospholipid bilayers that contain membrane proteins, carbohydrates, and other molecules. These membrane components contain ionizable functional groups such as the acidic and basic side chains of amino acids (3) that contribute to the net charge of the particle. Isoelectric focusing methods take advantage of this property to separate biological particles based on their pI.

The pH gradient in IEF is established by electrolysis of water, supported by anolyte and catholyte and stabilized by carrier ampholytes, or set by amphoteric species immobilized in a gel. The vast majority of IEF experiments are performed using immobilized pH gradients in a gel since this method provides superior reproducibility, linearity of the pH gradient, and many choices of broad to narrow gradients. However, the size of biological particles precludes the use of gels so free-solution IEF methods must be used.

In free-flow IEF, the pH gradient is established by application of an electric field across a separation chamber perpendicular to the flow of a solution through the chamber containing the sample and carrier ampholytes. Species migrate to their pI as they travel through the chamber, and can be collected as separate fractions as they exit the device. This technique has been used to achieve separations of organelles from cultured cells in a microfluidic device.2 The authors demonstrated several applications including the separation of mitochondria and nuclei, and the detection of differences in a functional property of mitochondria, the mitochondrial membrane potential. Free-flow IEF benefits from its speed and the possibility of continuously collecting fractions, which allows for higher-throughput separations.

Capillary IEF is a technique that achieves high-resolution separations with very small amounts of sample and low limits of detection. In this technique, a pH gradient is established by application of an electric field across a capillary containing the sample and carrier ampholytes, and is stabilized by buffer reservoirs containing anolyte (acid) and catholyte (base). Capillary IEF has been used to separate different single-celled organisms such as yeast and bacteria.3 In this work, the use of fluorescent internal standards allowed the authors to determine the pIs of the detected microorganisms. Capillary IEF has also been used to test the purity and measure the concentration of an extraction of virus particles.4 We recently used this technique to accurately determine the pIs of individual mitochondria from cultured cells.5 The use of internal standards and a mitochondrial-specific fluorescent probe allowed us to measure changes in the mitochondrial pI distribution upon mild treatment of the surface of these organelles with a protease.

New developments and refinements in microfluidic free-flow IEF and capillary IEF will lead to more exciting applications of these techniques to the analysis of biological particles. We are confident that these methods will be widely used in clinical and research settings in the future.

References
  1. Subirats, X.; Blaas, D.; Kenndler, E., Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organ- elles, and cells. Electrophoresis 2011, 32 (13), 1579-1590.
  2. Lu, H.; Gaudet, S.; Schmidt, M. A.; Jensen, K. F., A microfab- ricated device for subcellular organelle sorting. Anal. Chem. 2004, 76 (19), 5705-5712.
  3. Horka, M.; Ruzicka, F.; Horky, J.; Hola, V.; Slais, K., Capil- lary isoelectric focusing and fluorometric detection of proteins and microorganisms dynamically modified by poly(ethylene gly- col) pyrenebutanoate. Anal. Chem. 2006, 78 (24), 8438-8444.
  4. Horka, M.; Kubicek, O.; Kubesova, A.; Kubickova, Z.; Rosen- bergova, K.; Slais, K., Testing of the influenza virus purification by CIEF. Electrophoresis 2010, 31 (2), 331-338.
  5. Wolken, G. G.; Kostal, V.; Arriaga, E. A., Capillary Isoelectric Focusing of Individual Mitochondria. Anal. Chem. 2011, 83 (2), 612-618.