Gel electrophoresis

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

Principle

Different ion mobility is used in different electrophoresis methods for separating ionic substances in an electric field and, for example, be sent separately to a measurement.

In gel electrophoresis, migrates from a mixture to be separated, the electrically charged molecules under the influence of an electric field (see: electrophoresis) by a gel which is in an ionic buffer solution. Depending on the size and charge of the molecules, they move at different rates through the gel acting as a molecular sieve. In this case, small, negatively charged molecules (anions ) migrate fastest toward the positively charged anode and positively charged molecules (cations ) in the direction of the negatively charged cathode. The theories underlying the complementary Ogston sieve theory and the reptation theory. While the sieve theory ( for example, proteins or micelles ) describes the retention (synonym retention) of spherical macromolecules due to a defined porosity of the gel matrix, the reptation theory is a retention of macromolecules by friction non-spherical macromolecules in the gel matrix ( e.g., DNA and RNA).

Gel matrix

The polymer molecules of the gel form a more or less close-knit, three-dimensional network migration ( migration ) of the slow molecules to be separated in the electric field more or less.

Agarose

Agarose gels are relatively large pores (150 nm at one percent, 500 nm at 0.16 percent gels ) and are well suited for the separation of DNA and high molecular weight proteins. However, the main area of ​​application is the separation of nucleic acids.

Polyacrylamide

Gels of polyacrylamide prepared by polymerizing acrylamide. They have much smaller pores (3-6 nm). The pore size is dependent on the acrylamide concentration and the degree of crosslinking. Hereby proteins are often separated.

Implementation

The classic analysis by gel electrophoresis as a zone electrophoresis. A method for obtaining a higher resolution is the discontinuous electrophoresis.

In gel electrophoresis, heat is generated. These must be removed in order to ensure optimum conditions. Therefore, the gel should be performed in cooled apparatuses at constant temperatures in order to achieve reproducible results.

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

Evaluation

Same molecules run into discrete zones - colloquially referred to as gangs - through the gel. Several samples can be run parallel to each other at the same time by the same gel. Is the size of some molecules known, can be estimated by comparing the bands with the remaining bands of the size of other molecules. Such molecular weight standards are commercially available. Similarly operates a Komigrationsstandard with the aid of an unknown sample having a known composition is compared. As molecular mass standards DNA or proteins.

A determination of the amount of a substance in a gang or the relative proportion of a gang (see: quantification ) is possible after staining of the gel and subsequent densitometric analysis, with the restriction that not for very dark bands of the inner region of the band due to lack of exposure to light can be counted with. For the determination of the measured values ​​of a gel such as run lengths, molecular weights, or quantification evaluation normalization software is utilized in most cases.

For the evaluation of the gel after the electrophoresis, the molecules to be separated are radioactively labeled, either prior to electrophoresis, and then detected in a staggered or autoradiography after electrophoresis with different dyes.

Of nucleotides in the ethidium bromide is commonly used, the intercalated with nucleic acids, and this makes it visible under UV light. Proteins can be stained directly, eg with Coomassie brilliant blue or as part of the silver staining. An alternative to staining, the subsequent blotting. We distinguish:

  • Western blot ( immunological detection of proteins with labeled antibodies )
  • Southern blot ( DNA detection by hybridizing with DNA or RNA probes)
  • Northern blot (detection of mRNA by hybridization with nucleotide probes also )

Areas of application

Gel electrophoresis found in molecular biology, biochemistry and food analysis application. Gels can be produced even without much effort. Ready gels and the corresponding buffer systems can also be purchased commercially.

There are numerous special applications:

  • SDS-PAGE for the separation of mixtures of substances (often proteins ) as molecular size
  • IEF separation of proteins according to their isoelectric point
  • 2D gel electrophoresis, a combination of SDS-PAGE and IEF of complex protein mixtures
  • QPNC -PAGE for the analysis of metalloproteins
  • Native gel electrophoresis to study the protein folding
  • SDD -AGE for the investigation of protein aggregates
  • Pulsed -field gel electrophoresis ( PFGE) to separate large DNA fragments
  • Capillary electrophoresis ( CE)
Electrical mobility Gel electrophoresis#Agarose Intercalation (chemistry) QPNC-PAGE SDD-AGE Digital Object Identifier
364430
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