Gel electrophoresis

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Experimental setup
Vertical gel electrophoresis apparatus of SDS-PAGE
Horizontal gel electrophoresis apparatus for agarose gel electrophoresis

Gel electrophoresis (parts of the word: gel | electro | phoresis - the latter derived from ancient Greek φερειν pherein ' to carry') is an analytical method in chemistry and molecular biology to separate different types of molecules .

principle

The different ion mobility is used in various electrophoresis methods to separate ionic substances in an electric field and e.g. B. separately feed a measurement.

In gel electrophoresis, a mixture of electrically charged molecules to be separated migrates under the influence of an electric field through a gel which is in an ionic buffer solution ( electrophoresis buffer ). Depending on the size and charge of the molecules, they move at different speeds through the gel, which acts as a molecular sieve. Small, negatively charged molecules ( anions ) migrate fastest in the direction of the positively charged anode and positively charged molecules ( cations ) in the direction of the negatively charged cathode . The underlying theories are the complementary Ogston Sieve Theory and the Reptation Theory . While the sieve theory describes the retention of spherical macromolecules (e.g. proteins or micelles ) through a defined porosity of the gel matrix, the reptation theory deals with the retention of macromolecules by friction of non-spherical macromolecules on the gel matrix (e.g. DNA and RNA ).

Gel matrix

The polymer molecules of the gel form a more or less close-meshed, three-dimensional lattice that more or less slows down the migration (migration) of the molecules to be separated in the electric field.

Agarose

Agarose gels have relatively large pores (150 nm for one percent, 500 nm for 0.16 percent gels) and are well suited for separating DNA and high molecular weight proteins . The distance between DNA bands of different lengths depends on the concentration of agarose in the gel. Higher concentrations require longer run times (sometimes even days). However, the main area of ​​application is the separation of nucleic acids. Agarose gels are made from the natural polysacchardi polymers from seaweed . The electrophoresis with agarose gel is a physical setting. After the experiment, the result can be stored frozen using a plastic bag.

Polyacrylamide

Gels of polyacrylamide are by polymerization of acrylamide produced. They have much smaller pores (3–6 nm). The pore size depends on the acrylamide concentration and the degree of crosslinking. Often proteins between 5 and 20,000 kDA are separated with this.

Strength

Another possibility is the use of partially hydrolyzed potato starch in concentrations between 5% and 10%. It is a non-toxic medium for the electrophoresis of undenatured proteins. The separation takes place according to size and charge and the visualization is done by naphthol black or amido black coloring. Without the addition of biocides , starch gels tend to decompose microbially.

execution

Agarose gel with DNA and running dye in the gel pockets, before electrophoresis.

Classic gel electrophoresis is carried out as zone electrophoresis . One method of achieving higher resolution is batch electrophoresis .

Gel electrophoresis generates heat . This must be removed to ensure optimal conditions. Therefore, the gel electrophoresis should be carried out in cooled apparatus at constant temperatures in order to achieve reproducible results.

Ideally, electrophoresis is ended when the smallest or most mobile molecules have reached the end of the gel. This guarantees the highest possible separation of the molecules.

evaluation

Identical molecules run through the gel in discrete zones - colloquially known as bands . Several samples can run in parallel through the same gel. If the size of some molecules is known, one can estimate the size of the other molecules by comparing their bands with the remaining bands. Such molecular mass standards are commercially available. A comigration standard works similarly , with the help of which an unknown sample composition is compared with a known sample composition. DNA or proteins are used as molecular mass standards.

A determination of the amount of a substance in a band or the relative proportion of a band (see: Quantification ) is possible after coloring and photography or scanning of the gel and a subsequent densitometric evaluation, with the restriction that in the case of very dark bands the inner area of ​​the Band cannot be counted due to lack of light exposure. To determine the measured values ​​of a gel such as Evaluation software is used in most cases, for example, widths, molecular masses , quantifications or normalization.

DNA bands under UV light in an agarose gel stained with ethidium bromide.

To evaluate the gel after electrophoresis, the molecules to be separated are either radioactively labeled before electrophoresis and then detected in an autoradiography or mixed with various dyes after electrophoresis.

Ethidium bromide is often used in nucleotide analysis, which intercalates with nucleic acids and makes them visible under UV light . Proteins can be stained directly with protein dyes , e.g. B. with Coomassie brilliant blue or in the course of silver coloring . Subsequent blotting is an alternative to staining . One differentiates:

Areas of application

Gel electrophoresis is used in molecular biology , biochemistry and food analysis. Gels can be made by yourself without much effort. Finished gels and the corresponding buffer systems can also be purchased commercially.

There are numerous special applications:

literature

Web links

Commons : gel electrophoresis  - collection of images, videos and audio files

Individual evidence

  1. ↑ Alexander George Ogston : The spaces in a uniform random suspension of fibers. In: Transactions of the Faraday Society. Vol. 54, 1958, ISSN  0014-7672 , pp. 1754-1757, doi : 10.1039 / TF9585401754 .
  2. ^ Gary W. Slater, Jean Rousseau, Jaan Noolandi, Chantal Turme, Marc Lalande: Quantitative analysis of the three regimes of DNA electrophoresis in agarose gels. In: Biopolymers. Vol. 27, No. 3, 1988, ISSN  0006-3525 , pp. 509-524, PMID 3359012 , doi : 10.1002 / bip.360270311 .
  3. ↑ Oscar J. Lumpkin, Philippe Déjardin, Bruno H. Zimm: Theory of gel electrophoresis of DNA. In: Biopolymers. Vol. 24, No. 8, 1985, pp. 1573-1593, PMID 4041551 , doi : 10.1002 / bip.360240812 .
  4. ↑ Agarose Gel Electrophoresis. Retrieved February 18, 2015
  5. ^ Joseph Sambrook, David Russell: Molecular Cloning - A Laboratory Manual
  6. ↑ Gordon AH: Electrophoresis of proteins in polyacrylamide and starch gels . American Elsevier Publishing Company, Inc, New York 1975.
  7. ↑ Smithies O .: Zone electrophoresis in starch gels: group variations in the serum proteins of normal adults . In: Biochem. J. Band 61 , no. 4 , 1955, pp. 629-641 , PMID 13276348 , PMC 1215845 (free full text).