Cellular Biology

The Basics of Gel Electrophoresis

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One of the workhorse techniques widely utilized in the study of biological macromolecules (DNA, RNA, protein) is gel electrophoresis. Electrophoresis is the process of inducing the migration of charged particles under the influence of an electrical field or current. Gel electrophoresis combines the application of electrical current with a supporting media or gel matrix, which acts like a sieve to separate molecules according to their unique properties. One-dimensional electrophoresis separates molecules in a complex mixture by either their charge or their size (molecular weight). Two-dimensional electrophoresis separates molecules according to both the charge (or pH) and relative size of particles. How particles migrate in a gel can be influenced by manipulation of the gel matrix, the buffer and the electrical field applied to the system.

Matrices can vary but must be mechanically stable and have uniform, but adjustable pore sizes. A suitable gel matrix must also be chemically inert, so that only the electrical current and buffering system is impacting the movement of charged sample. Two widely and commonly used gel matrices are agarose and polyacrylamide.

Agarose is extracted from red seaweed, that yields gels with a large pore size to analyze macromolecules over 10 nanometers in diameter. Agarose gels are the standard for use in the analysis of nucleic acids and large protein aggregates. Agarose gels are prepared in as much the same way as edible gelatin: the agarose is dissolved in boiling water and then allowed to cool in a slab mold, solidifying into the desired size, width and shape. Polyacrylamide is a stable, transparent gel that results from inducing the cross-linking of multiple monomers of acrylamide in solution. Un-polymerized acrylamide monomer (powder or in solution) is very toxic and should be handled with care. Poly-Acrylamide Gel Electrophoresis, or PAGE, is a commonly used matrix for the separation of proteins and other molecules requiring a narrow pore size and high resolution.

Electrophoretic separation is done in a buffer having a defined pH, a high buffering capacity and low, constant ionic strength. This is to assure that as many sample ions as possible are charged (positively or negatively) and maintain their charge throughout migration in one direction. A buffer of low ionic strength allows sample ions to contribute to the total current, which also facilitates their movement. Adjusting the volume, pH or salt concentration of the buffer system can dramatically influence ion conductivity in a gel run.

During electrophoresis, buffer ions are carried through the gel along with sample ions. This should be done with as little energy as possible to avoid generating too much friction heat in the system. This consideration must be balanced against attaining enough field strength in the buffer (electrical current) necessary to induce electrophoretic migration. Smaller molecules will migrate within a matrix faster than larger molecules of the same charge along an electrical current. Molecules will settle along a matrix in a predictable pattern of larger (slow moving) particles at the top of gel and subsequently smaller (fast moving) particles settling towards the bottom edge. Care must always be taken not to run a gel so long or so fast that small molecules move out of the matrix and diffuse into the surrounding buffer.

Reference: Electrophoresis in Practice: A guide to Methods and Applications of DNA and Protein Separations, 4th Ed., 2005, Westermeier R., p406

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