Discontinuous electrophoresis

The discontinuous electrophoresis is a biochemical method for the separation of biomolecules . It was first described in 1964 by L. Ornstein and BJ Davis.

principle

Batch electrophoresis uses two different buffers and, compared to continuous electrophoresis, produces a high level of definition. It is used as polyacrylamide gel electrophoresis with a separating gel and a front of it stacking used. The discontinuity relates to different pH values ​​of the buffers (both the gel buffer and the electrode buffer) as well as the different pore sizes of the separating gel and the stacking gel. The decisive factor here is the discontinuity in the pH value. The pH value can be freely selected, the associated buffer components can be found in an online database. Here, the gel buffer contains a link Sammelgel- p fully ionized H quickly movable ion (gamma) and a partially ionized, thus moving slowly (alpha), with a common counter-ion (beta). The mobility of gamma is greater, that of alpha less than that of proteins. Under these conditions, when a voltage is applied, a stack of ions forms, for which Kohlrausch's regulating function applies:

${\displaystyle K_{E}=\sum _{j=1}^{n}{\frac {c_{j}}{u_{j}}}}$

Here, the persistent constant , n the number of ions present, and the mobility and concentration of the j th ion with ${\displaystyle K_{E}}$ ${\displaystyle u_{j}}$ ${\displaystyle c_{j}}$

${\displaystyle u_{j}={\frac {A\cdot d_{j}\cdot \zeta }{I\cdot t}}}$.

Here, A the cross section, the conductivity, I the current flowing and by the j th ion in the time t distance traveled. ${\displaystyle \zeta }$ ${\displaystyle d_{j}}$

This stack formation can be followed very nicely if one subjects commercially available, pre-colored molecular mass standards to the electrophoresis, a stack of closely spaced bands is formed, which moves at constant speed through the stacking gel. The separation can also only be carried out in the stacking gel, this process is called isotachophoresis . In a stack of three components abc, the bands of a and b as well as b and c partially overlap, but not the bands of a and c.

At the boundary between stacking gel and separating gel, the pH value suddenly changes , so that the only partially ionized ion alpha is completely ionized, its mobility increases and it overtakes the protein stack. From then on, the proteins move in a constant electric field; their speed depends on their charge and size. In contrast to isotachophoresis , where the entire stack moves at the same speed and the separation no longer improves after the stack formation, the distance of the protein bands increases with the distance in the separation gel.

Under suitable conditions it can be achieved that the separation in the separation gel is determined either only by the charge or only by the size. Separation according to charge (in a very open-pored matrix) is only rarely carried out in DISK electrophoresis; isoelectric focusing is more suitable here .

In contrast, the proteins can be treated with denaturing, anionic detergents such as sodium dodecyl sulfate (SDS) or with cationic detergents such as cetyltrimethylammonium bromide ( CTAB ) or 16-BAC prior to electrophoresis . One then speaks of SDS-PAGE according to Laemmli or CTAB-PAGE or BAC-PAGE . Most proteins bind a constant amount of detergent (1 molecule of SDS per 3 amino acids or 1.4 g / g), which means that all proteins have a constant charge / weight ratio. They all experience the same acceleration in the electrical field, but are slowed down by the gel depending on their size. Overall, the separation takes place according to the molecular radius ( not, as is often claimed, according to the molecular mass). The migration speed is described by the Ferguson equation: where T is the gel concentration (total = crosslinker + acrylamide) and and regression parameters . This equation enables the molecular radius of a protein to be determined by comparing the electrophoretic mobility with that of standard proteins. Often this is only done with one gel concentration, but accuracy suffers. ${\displaystyle \log {(M)}=\log {(M_{0})}-K_{R}\cdot T}$ ${\displaystyle M_{0}}$ ${\displaystyle K_{R}}$

The SDS-PAGE is the older and far more common method used, CTAB-PAGE are glycoproteins but sharper bands. Glycoproteins contain negatively charged sugar residues, the number of which is very variable and thus also the speed of migration in the electric field. In CTAB-PAGE, this heterogeneity is balanced out by the ionic binding of additional detergent molecules. The native PAGE is used to preserve the native protein fold upon separation .

literature

• Hans Rainer Maurer : Disc electrophoresis and related techniques of polyacrylamide gel electrophoresis , Verlag de Gruyter, Berlin, New York 1971, 1. engl. Edition, ISBN 978-3-11-003495-0 .
• M. Niepmann: Discontinuous native protein gel electrophoresis: pros and cons. In: Expert review of proteomics. Volume 4, Number 3, June 2007, pp. 355-361, doi : 10.1586 / 14789450.4.3.355 , PMID 17552919 .
• D. Wheeler, A. Chrambach, P. Ashburn, TM Jovin: Discontinuous buffer systems operative at pH 2.5-11.0, 0 degrees C and 25 degrees C, available on the Internet. In: Electrophoresis. Volume 25, Numbers 7-8, April 2004, pp. 973-974, doi : 10.1002 / elps.200305799 , PMID 15095436 .

Individual evidence

1. ↑ L. Ornstein, BJ Davis: Disc Electrophoresis -1. Background and Theory. In: Ann NY Acad Sci. Volume 121, 1964, pp. 321-349.
2. ↑ Archive link ( Memento of the original from January 25, 2017 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice.