Electrophoresis

Electrophoresis with DNA fragments obtained by PCR ;
(1) is the father,
(2) the child,
(3) the mother.

Electrophoresis (outdated cataphoresis ) describes the migration of charged colloidal particles or dissolved molecules through an electric field .

The pioneer of electrophoresis was Arne Tiselius (1937). The breakthrough came after Oliver Smithies found in 1955 that starch gels were very suitable for electrophoresis (later largely replaced by acrylamide, for example).

description

The drift speed (also “migration speed ”) of the colloidal particles, typically proteins or nucleic acids, in electrophoresis is proportional to the field strength and the ion charge , inversely proportional to the particle radius and the viscosity of the substance. In addition, the ionic environment of the solution, in which electrical current flows, plays an important role. The physical cause of the movement is the shear force in the electrical double layer that surrounds the colloid ( star double layer ) and sets the charged liquid in relative movement to the macromolecule. In gel electrophoresis , the ratio between the particle radius and the pore size of the gel used as the carrier medium also plays a role, because the gel acts as a molecular sieve , so that a larger particle radius has a more inhibiting effect on the migration speed than would be expected from viscosity alone . Due to the different ion charge and the particle radius, the individual substances (molecules) move at different speeds through the carrier material and achieve separation according to their electrophoretic mobility . This makes electrophoresis very suitable for separating mixtures of substances (especially mixtures of molecules). Liquids, gels (see gel electrophoresis , mostly with polyacrylamide or agarose ) or solids can be used as carrier material . ${\displaystyle v}$ ${\displaystyle E}$ ${\displaystyle Q}$ ${\displaystyle r}$ ${\displaystyle \eta }$

Agarose gels are mainly used for the separation of DNA fragments, while proteins are mostly separated in polyacrylamide gels . The methods used for proteins are SDS-PAGE and Western Blot . Proteins have as zwitterions with additional charges by a detergent such as sodium dodecyl sulfate ( English sodium dodecyl sulfate , SDS) are charged to a separation according to the heterogeneous charge densities to a separation according to the molecular weight to come. By adding SDS and boiling ( denaturing ) the proteins adsorb the aliphatic end of the negatively charged sodium lauryl sulfate proportionally to their unfolded length (and also proportionally to the molecular mass). About 1.4 grams of SDS bind per gram of protein in one percent SDS solutions. The negatively charged sulfate groups of the SDS molecules repel each other, which promotes the unfolding (linearization) of the proteins, provided the protein does not have any disulfide bridges. Therefore, when determining the molar mass, reducing agents are added to convert the disulfides into thiols . Since several hundred negatively charged SDS molecules bind to the protein molecules, the intrinsic charge of the proteins in the basic pH of the gel can be neglected.

Electrophoretic Mobility

Two SDS gels after the end of the sample run and staining of the protein bands with Coomassie

The electrophoretic mobility of two particles to be separated must be different in order to achieve separation by means of electrophoresis. Electrophoretic mobility is the sum of many physical factors that ultimately affect the rate at which a particle migrates during electrophoresis. The general driving force that causes the movement of the particles is the force that acts on a particle with a certain charge within an electric field with a given field strength . ${\displaystyle F}$ ${\displaystyle q}$ ${\displaystyle E}$

${\displaystyle F=q\cdot E}$

This is counteracted by a force that results from the viscosity and the size of the particle (idealized for spherical particles:) and can be calculated according to Stokes' law . ${\displaystyle \eta }$ ${\displaystyle 6\cdot \pi \cdot r}$

${\displaystyle F=6\cdot \pi \cdot r\cdot \eta \cdot v}$

Theoretical electrophoretic mobility results from these two equations . Theoretically for the reason that these two equations only apply to an idealized, carrier-free state with an infinitely dilute (practically salt-free, which, however, contradicts the principle of electrophoresis, since salt ions are required as mobile charge carriers) electrolytes . It is also assumed that the accelerating force corresponds to the frictional force and therefore a constant migration speed prevails. Therefore, the ion mobility in this model is as follows: ${\displaystyle \mu _{e,p}^{0}}$

${\displaystyle \mu _{e,p}^{0}={\frac {v}{E}}={\frac {q}{6\cdot \pi \cdot r\cdot \eta }}}$

In real systems there are further factors such as the friction between the hydration shells ( electrophoretic effect ), the deformation of the charge distribution as relaxation in the electric field ( dissipative effect , see ion atmosphere ), the degree of dissociation of the electrolyte and effects from the carrier material ( molecular sieve , electroosmosis and Adsorption effects ) to wear.

While traditional theories assume that the electrophoretic activity of a particle requires a net charge on the particle, new results from molecular dynamics simulations suggest that, due to the molecular structure of water on the surface, even uncharged particles can show electrophoretic activity.

Offord empirically found the following relationship between mobility and net charge and molar mass: ${\displaystyle Z}$

${\displaystyle \mu ={\frac {Z}{M^{2/3}}}}$

warmth

The maximum voltage that can be applied is limited by the heating of the gel, which is caused by the friction effects of the migrating molecules. The heat can lead to uneven migration of the molecules, increases diffusion (resulting in blurred bands) and can denature the molecules . The heat generation is determined by the applied voltage and the electrical conductivity of the system used, in particular the conductivity of the electrophoresis buffer .

species

• Affinity electrophoresis
• Agarose gel electrophoresis
  • SDD-AGE
• Density gradient electrophoresis (carrier-free electrophoresis)
• Discontinuous electrophoresis
• Electro-osmosis occurs in electrophoretic processes
• Electrofocusing
• Free-flow electrophoresis
• Gel electrophoresis
  • 2D gel electrophoresis (two-dimensional electrophoresis)
• Gradient electrophoresis
• Dielectrophoresis
• Immunoelectrophoresis
• Isoelectric focusing occurs in electrophoretic processes
• Capillary electrochromatography
• Capillary electrophoresis
• Lipid electrophoresis
• Western blot
• Isotachophoresis
• Polyacrylamide gel electrophoresis
  • BAC-PAGE
  • CTAB-PAGE
  • Native PAGE
  • QPNC-PAGE
  • SDS-PAGE (SDS polyacrylamide gel electrophoresis)
• Pulsed field gel electrophoresis
• Serum electrophoresis
• Migration electrophoresis
• Zone electrophoresis

application

Electrophoresis is mainly used as an analytical method in biology and medicine. The most important applications include serum electrophoresis , as well as DNA analysis in the form of fragments and DNA sequencing. Here the possibility is used to separate molecules of different lengths from one another. To determine the measured values ​​of a gel such as Specialized evaluation software is used, for example, widths, molar masses, quantifications or normalization. Electrophoresis also forms the basis for the separation of proteins and for the high-tech processes of proteome research. The graph of the results is an electropherogram . In addition to the analytical methods, preparative electrophoresis methods (including free-flow electrophoresis ) are used to obtain milligram quantities of purified proteins .

Other technical applications:

• Electronic paper
• Cathodic dip painting
• Electrofiltration

history

Electrophoretic effects were first investigated in 1807 by Pjotr ​​Iwanowitsch Strachow and Ferdinand Friedrich von Reuss . Electrophoresis was developed by Arne Tiselius in 1937 as a method with which colloids in a carrier liquid could be separated in an electric field ( English moving boundary electrophoresis , electrophoresis with a moving boundary layer ). Tiselius received the Nobel Prize in Chemistry for this in 1948 . In the 1940s, solid phases were increasingly used for better separation ( English zone electrophoresis , zone electrophoresis), such as the starch gel from Oliver Smithies or filter paper . Since these tend to decompose microbially , other hydrogels have also been used, e.g. B. agarose or polyacrylamide . While radial electrophoresis was often carried out on round disks in the 1950s ( English radial electrophoresis , disk electrophoresis), nowadays rectangular gels are used almost exclusively ( English slab gel electrophoresis , gel plate electrophoresis).

literature

• Manfred H. Gey: Instrumental Analytics and Bioanalysis . 3. Edition. Springer, Berlin / Heidelberg 2015, ISBN 978-3-662-46254-6 , Chapter 8: Electrophoresis , doi : 10.1007 / 978-3-662-46255-3_8 .
• RE Offord: Electrophoretic mobilities of peptides on paper and their use in the determination of amide groups. In: Nature. Volume 211, Number 5049, August 1966, pp. 591-593. PMID 5968723 .

Individual evidence

1. ↑ Entry on electrophoresis . In: IUPAC Compendium of Chemical Terminology (the “Gold Book”) . doi : 10.1351 / goldbook.E02022 .
2. ↑ V. Knecht, HJ Risselada, AE Mark, SJ Marrink: Electrophoretic mobility does not always reflect the charge on droplet of oil . In: Journal of Colloid and Interface Science . tape 318 , no. 2 , January 15, 2008, p. 477-486 , doi : 10.1016 / j.jcis.2007.10.035 ( PDF [accessed January 5, 2010]).
3. ↑ Reinhard Kuhn: Capillary Electrophoresis: Principles and Practice. Springer Science & Business Media, 2013, ISBN 978-3-642-78058-5 , p. 80.
4. ^ Reuss, FF: Sur un nouvel effet de l'électricité galvanique . In: Mémoires de la Société Impériale des Naturalistes de Moscou . tape II , 1809, p. 327-337 .
5. ↑ Arne Tiselius: A new apparatus for electrophoretic analysis of colloidal mixtures . In: Transactions of the Faraday Society . tape 33 , 1937, pp. 524-531 , doi : 10.1039 / TF9373300524 .
6. ↑ O. Smithies: 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).