What Is Electrophoresis & How Does it Work?
Electrophoresis is a laboratory technique used to transport charged protein molecules and nucleic acids through a solvent using an electrically-charged field. It is considered a simple, rapid, and sensitive analytical technique.
Numerous biological molecules carry a net charge at any pH level other than their isoelectric point and migrate at a rate in proportion to their charge density.
A molecule’s mobility through an electric field depends on the following:
- Field strength
- The net charge on the molecule
- Size and shape of the molecule
- Ionic strength
- Properties of the matrix, such as pore size and viscosity
Different types of matrices are used in electrophoresis, including polyacrylamide and agarose. They are both a type of gel. As gel matrices, polyacrylamide and agarose offer a porous media that essentially behaves as a filter.
While polyacrylamide has a small pore size and is well suited for separating a large number of proteins and smaller nucleic acids, agarose has larger pore sizes, making it ideal for separating larger macromolecules, such as protein complexes and large nucleic acids.
Multiple forms of gel electrophoresis are used to separate nucleic acids and proteins, including polyacrylamide gel electrophoresis (PAGE). This form alone has several varieties, which can provide various information about specific proteins of interest.
For example, denaturing and reducing sodium dodecyl sulfate PAGE (SDS-PAGE) with a discontinuous buffer system is the most widely used electrophoresis technique and separates
proteins primarily by mass. Nondenaturing PAGE, also called native-PAGE, separates proteins according to their mass/charge ratio. Two-dimensional (2D) PAGE separates proteins by native isoelectric point in the first dimension and by mass in the second dimension.
Once a protein or nucleic acid is separated by electrophoresis, it can be detected in the gel matrix using different stains. They are then transferred onto a membrane for detection by western blotting or analyzed using mass spectrometry. For this reason, DNA and protein gel electrophoresis is an essential step in proteomics.