Page 148 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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376 Electrophoresis

different salt concentrations and pH have been used to trophorsing the slab for a period before adding the pro-
produce stacking of native proteins by varying their rela- tein. This pre-electrophoresing settles the ampholines at
tive charges in a plane in the gel. This changes the relative their varied isoelectric points. Because ampholines con-
1
mobilities, so that T ± /C for each protein is not equal to tain many functional groups, they possess greater buffer-
that in the next plane (called isotachophoresis). ing capacity than a similar mass of peptides, and so the
Procedures similar to those used for proteins can be gradient is not disturbed by the presence of the protein.
used for nucleic acids. Here, the gels not only act as sta- In these experiments the current is very low after the
ble supports for mechanical handling, but also as separate initial removal of excess charged diffusible ions because
mixtures according to mass. No additional detergents are the only remaining transportable ions in the system are H +
−
required because the structure of nucleic acids in solution and OH . The focused boundaries follow the approximate
is essentially a random coil formed from highly charged shape described by Eq. (21) and are stationary within the
polyelectrolytes. gel for long periods. True equilibrium, where the position
For technical reasons gels containing less than 2% (by is independent of very long times, is rarely achieved be-
−
+
weight) of acrylamide are unmanageable, and yet even cause the transport of the H and OH continues, and this
at that concentration the pore sizes are too small to admit slowly drags the ampholines with them, eventually de-
large macroions (radii >10 nm). To handle these ions, gels stroying the gradient. Ampholines have been developed
must be formed from polysaccharides. The most common that can be copoymerized into the polyacrylamide gel to
is agarose, a polymer of galactose. Agarose is a fraction prevent their movement, and when these are used the pH
from agar, a seaweed polysaccharide, which is partly sul- gradient is formed mechanically before the acrylamide is
fonated so the charged parts must be removed for use in polymerized.
electrophoresis. These gels are mechanically fragile but With this technique it is possible to focus individual pro-
can be partly stabilized if a few covalent bonds are formed teins from a mixture into bands that are fractions of a mil-
between some of the galactose units. limeter wide and have effective concentrations exceeding
the solubility of the protein. At these high concentrations
the protein “steals” the water from the polyacrylamide
D. Isoelectric Focusing and Isotachophoresis
chains, and this weakens the matrix, making the columns
The procedures described for PAGE employ the charges fragile at this point. The technique can be applied to the
only as a means of electrically driving the macroions along study of native or denatured proteins. In the latter case a
the gel in a fixed direction in order to separate mixtures charged detergent (e.g., SDS) is displaced from the protein
and estimate relative masses. These procedures yield lit- during electrophoresis because the unassociated detergent
tle information on the charge of the protein or utilize moves to the anode, therefore forcing the micelles to dis-
the unique pH where polyampholytes have no net charge sociate in order to maintain chemical equilibrium between
(see Section II.D) in order to separate a complex mix- associated and free detergent. Eventually, all the adsorbed
ture. A stationary boundary forms at this pH, but since SDS is stripped away from the peptide, leaving it with its
the object of the experiment is to both separate and con- native intrinsic charge (and possible insolubility).
centrate proteins into narrow bands, a stable pH gradient For preparative procedures where relatively high con-
must be generated. To do this the solution containing the centrations of a mixture of polyampholytes are applied
monomeric acrylamide must contain ampholyte buffers initially to the pH gradient, it is often uneconomic to em-
that when electrophoresed move more rapidly than the ploy supporting ampholines in the gel. In these cases a pH
polyampholytes and settle at their respective isoelectric gradient is generated during preparation of the gel column
points where they buffer the pH. If a wide-ranging mixture using conventional buffers. The object of an experiment
of ampholytes, themselves having many different isoelec- is to isolate one polyampholyte selectively by stacking it
tric points, is used, the result is a stable pH gradient, which at an interface between two zones using the Kohlrausch
can be made approximately linear with distance between regulating function as an underlying theoretical guide (see
the electrodes. Section I.A). In order to apply the technique it is neces-
A pH gradient can be formed if water is electrolyzed— sary to have studied the protein using analytical PAGE in
acid at the anode and alkaline at the cathode—but the order to ascertain its relative charge and mass. The latter is
buffering capacity of water is negligible and the gradient required in order to adjust the properties of the supporting
is easily swamped by the protein when they are included. polyacrylamide gel in isotachophoresis so that it does not
To make a stable gradient a variety of organic ampholines
1 They can be likened to polyacrylic acid, where each monomer ex-
have been synthesized, with various proportions of acidic
poses a charged carboxylic group, but in the ampholines each monomer
and basic groups in the heterogeneous mixture of ampho-
exposes a basic and an acidic group, so by producing a mixture of
lines. When these are included in the polyacrylamide gel oligomers from the ampholines a wide range of isoelectric points can
as free solutes, a pH gradient can be generated by elec- be produced.