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Gel electrophoresis


SDS-PAGE autoradiography

Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. Gel Electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules to use other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.

The first part, "gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked 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 different 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. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning.

The second part, "electrophoresis", refers to the electromotive 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, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative). 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-phosphate backbone. Double-stranded DNA fragments natually behave as long rods, so their migration through the gel is relative to their radius of gyration, 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 hydroxide or formamide, are used to renature 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 denatured in the presense of a detergent such as sodium dodecyl sulfate/sodium docecyl phosphate (SDS/SDP) 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 overall 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 stained to make them visible. Ethidium bromide, Silver, or Coomassie 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 luminesce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel (as in the example shown above).

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. The distance a band travels is approximately inversely proportional to the log10 of the size of the molecule.

Types

Gel electrophoresis is used in genetics and biochemistry:

See also

External links


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