Home > Gel electrophoresis

The first part of the term, Gel, refers to the matrix used to separate the molecules. In most cases the gel is a cross-linked polymer whose porosity can be controlled by the scientist. When separating proteins or small nucleic acids ( DNA, RNA, or oligonucleotides ) the gel is usually made with differfent concentrations of acrylamide and a cross-linker , producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids ((greater than a few hundred bases , the preferred matrix is purified agarose. In both cases, the gel forms a solid but porous matrix that looks and feels like clear jello. Polyacrylamide is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning.

The second part of the term, Electrophoresis, refers to the electro-motive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and then applying an electric current, the molecules will be moved through the gel at different rates, from the anode towards the cathode. In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar- phosphateIn chemistry, a phosphate is a polyatomic ion or radical consisting of one phosphorus atom and four oxygen. In the ionic form, it carries a -3 formal charge, and is denoted PO3-. In a biochemical setting, a free phosphate ion in solution is called inorgan backbone. Double-stranded DNA fragments natually behave as long rods, so their migration through the gel is relative to their radius of gyrationThe radius of gyration of an area with respect to a particular axis is the square root of the quotient of the moment of inertia divided by the area. It is the distance at which the entire area must be assumed to be concentrated in order that the product o, or, roughly, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds , such as Sodium hydroxideSodium hydroxide ( Na OH), also known as caustic soda or lye in North America, is a caustic metallic base used in industry (mostly as a strong chemical base) in the manufacture of paper, textiles, and detergents. Sodium hydroxide is occasionally used in t or formamide , are used to denature the nucleic acids and cause them to behave as long rods again.

Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are ususally denaturedIn biochemistry, a compound that has been denatured has lost its native state, or in other words, no longer has the shape that is most compact and allows for optimal biological activity. In proteins, a denatured protein has no secondary structure other th in the presense of a detergentA detergent is a compound, or a mixture of compounds, whose molecules have two distinct regions—one that is hydrophilic, and dissolves easily in water, and another region that is hydrophobic, with little (if any) affinity for water. As a consequence, thes such as sodium docecyl phosphateSodium dodecyl sulfate ( SDS or NaDS) ( C H(CH) O SO Na) (FW 288. 38), also known as sodium lauryl sulfate ( SLS), is an ionic detergent that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening ef (SDS) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein, so that the resulting denatured proteins have an overally negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like they were long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

After the electrophoresis run, when the smallest molecules have almost reached the anode, the molecules in the gel can be stainedStains are used in biological and medical disciplines to highlight cell structures and organelles, cells, or tissues for viewing, usually with an instrument such as a microscope. In vitro staining is the process of dyeing living tissues (compare with in v to make them visible. Silver or Coumassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules luminesceLuminescence is any emission of electromagnetic radiation. Historically, even radioactivity was first thought of as a form of as "radioluminescence", although it is today considered to be radioactivity since it involves more than electromagnetic radiation under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

Bands in different lanes that end up at the same "height" contain molecules that passed through the gel with the same speed, which usually means they are about the same size. There are special markers available, which contain molecules of known sizes. If such a marker was run on one lane in the gel parallel to the mixture(s), the bands it displays can be compared to those of the mixture(s) in order to determine their size.