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Gel electrophoresis is used to separate macromolecules like DNA, RNA and proteins. DNA fragments are separated according to their size. Proteins can be separated according to their size and their charge (different proteins have different charges). A solution of DNA molecules is placed in a gel. Because each DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules. The result is a series of ‘bands’, with each band containing DNA molecules of a particular size. The bands furthest from the start of the gel contain the smallest fragments of DNA. The bands closest to the start of the gel contain the largest DNA fragments. When is gel electrophoresis used to separate DNA fragments?Gel electrophoresis can be used for a range of purposes, for example: When is gel electrophoresis used to separate proteins?Thanks to TV shows like CSI, many people are familiar with the use of gel electrophoresis to separate macromolecules like DNA. However, gel electrophoresis can also be used to separate out proteins. Different proteins have different sizes, mainly due to the number of amino acid building blocks in their structure. Chemical modifications attached to the protein also affect its size. Different proteins also have different charges. This can result from both the types of amino acid used to construct them, as well as the types of modifications attached to them. Different types of electrophoresis gels are used to provide different types of information. The type of gel you choose therefore depends on the type of question you are asking. Size SeparationTypically, gels made from polyacrylamide are used to separate proteins on the basis their different sizes. Usually, the proteins are first treated with heat and a chemical called SDS in order to unravel the protein. SDS is a detergent that gives all the proteins the same overall negative charge so that when an electric current is applied to the gel, separation is only due to the size of the protein. This technique is called SDS-PAGE (SDS-Polyacrylamide gel electrophoresis). Small protein molecules move more quickly through the gel than larger proteins, resulting in a series of ‘bands’. Each band contains a protein of a particular size. These can be compared with standards of known sizes. An SDS-PAGE gel has been used to separate proteins on the basis of size. The samples are the blood of various shark species. The first lane contains markers of known sizes. Large proteins are at the top of the gel and small proteins are at the bottom. This technique might be used for many purposes, including purifying a particular protein, for example to isolate an enzyme for the food industry. Charge and pH separationIsoelectric focusing (IEF) and agarose gel electrophoresis are two ways that proteins can be separated by their different electrical charges. Unlike SDS-PAGE, the proteins are usually kept in their native (folded) state. The type of gel that is used, and the solution around the gel, are also different. In agarose gel electrophoresis, proteins are loaded in the middle of the well. Those with a strong negative charge move fastest towards the positive side of the gel, whereas positively charged proteins move in the opposite direction. This technique might be used to separate proteins that have the same molecular weight but different charges, or when size is not important (e.g. to look at changes in the presence of different protein during the development of a disease). Two-dimensional electrophoresisThese days, charge (IEF) and size (SDS-PAGE) separation are often employed together in two-dimensional electrophoresis, where charge separation is first used, and then these separated proteins are separated on the basis on size. This is a very effective method for identifying a particular protein from a tissue that may contain thousands of proteins and where there may only be small differences between control and treated samples (e.g. to look for a protein involved in resistance to insect predation in plants).
Simple laws of physics dictate that when current is applied to a medium containing charged species, those species will migrate towards the opposite charge. Depending on the medium through which they are moving, other characteristics – such as the size of the species present – can impact their movement, leading to separation. This is the basis on which electrophoresis techniques, such as agarose gel electrophoresis, are built – techniques that are widely used across the life sciences. What is electrophoresis? In this article, we will consider how agarose gel electrophoresis works, how it can be interpreted and some of its purposes. Electrophoresis is a technique that uses electrical current to separate DNA, RNA or proteins based on their physical properties such as size and charge. What is agarose gel electrophoresis?Agarose gel electrophoresis is a form of electrophoresis used for the separation of nucleic acid (DNA or RNA) fragments based on their size. Negatively charged DNA/RNA migrates through the pores of an agarose gel towards the positively charged end of the gel when an electrical current is applied, with smaller fragments migrating faster. The resulting bands can then be visualized using ultraviolet (UV) light.
How does gel electrophoresis work?Agarose is a component of agar. It forms a 3D gel matrix of helical agarose molecules in supercoiled bundles held by hydrogen bonds, with channels and pores through which molecules are able to pass. When heated, these hydrogen bonds break, turning the agarose to liquid and allowing it to be poured into a mold before it resets (Figure 1).
DNA gel electrophoresis steps, the gel electrophoresis machine, electrophoresis buffer and electrical separationThere are a number of key steps4 involved in choosing, setting up, running and analyzing agarose gels that we will now consider. 1. Determine the required gel percentage – 0.7–1% agarose gel is typically adequate for most applications, but it is important to choose a percentage appropriate for your samples and expected fragment sizes. Combine the agarose powder with the same buffer type to be used to run the gel and heat to melt the mixture, avoiding boiling. Tris-acetate-ethylenediaminetetraacetic acid (EDTA) (TAE) or tris-borate-EDTA (TBE)5 are often the buffers of choice, as tris-acid solutions are effective buffers for slightly basic conditions, keeping DNA deprotonated and soluble in water. The EDTA, a chelating agent, inactivates nucleases that may damage the DNA being analyzed.
How to read gel electrophoresisAgarose gels may be visualized on a UV light box in a dark room or using a self-contained light box linked to a camera. Whichever system is utilized, UV light is shone through the gel from below and bands of DNA fluoresce thanks to the intercalating dye bound to them. This may be captured using a camera with a specialized UV filter for your records. Marker ladders come with a guide to indicate the size of each band they include. By comparing this to bands in sample lanes, the sizes of the bands can therefore be determined. The relative amount of DNA between samples may also be compared, as higher DNA concentrations will produce brighter bands. An example is shown in Figure 4.
What is the purpose of gel electrophoresis?There are a number of reasons why the separation of DNA fragments may be desirable, many of which are widely applicable across the life science disciplines. Let’s consider some common purposes.
DNA agarose gel electrophoresis glossary
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