Electrophoresis is the process by which DNA, RNA, and proteins are separated according to their size. Negatively charged nucleic acids or proteins migrate towards a positive electrode at a speed relative to their size, with smaller fragments traveling faster, and ultimately further. Various electrophoresis techniques are used in biological research. These include horizontal and vertical gel electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis, and SDS-PAGE. Numerous additives are required to carry out electrophoresis successfully.
Molecular standards are essential in determining the approximate size of the macromolecules being analyzed. There are a wide range of DNA, RNA and protein ladders available in a range of sizes that can be used on either polyacrylamide or agarose gels.
Loading dyes and stains are both fundamental components of electrophoresis. Loading dyes are added to the nucleic acid or protein sample to increase the density of the sample and prevent it from diffusing into the buffer. Loading stains visualize the bands following electrophoresis, enabling analysis. The most commonly used loading stain for visualizing DNA/RNA is ethidium bromide, which fluoresces under UV light. Coomassie Brilliant Blue Dye is routinely used to visualize proteins.
Buffers are another essential addition to an electrophoresis experiment. Buffers ensure that the pH remains stable so that proteins or nucleic acids are not denatured. Furthermore, buffers provide the necessary ions for maintaining a constant current. Frequently used buffers include TBE (tris-borate-EDTA) and TAE (Tris-acetate-EDTA). When selecting a buffer for your experiment, it is necessary to consider various properties of the buffer, such as the molecular size, formal charges, and pKa value.
Detergents are crucial components of the electrophoretic separation of proteins. Detergents induce unfolding of proteins and provide a negatively-charged coating which enables migration and separation. Sodium dodecyl sulfate (SDS) is a commonly-used detergent that is available in a range of concentrations.