Page 146 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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374 Electrophoresis

polymerization by one of a variety of procedures, all of ported with the ion (or remain stationary on the gel), there-
which produce free radicals in solution. These radicals fore making their radii different from that expected from
are produced by hydrolysis of water, either photochem- crystallographic models. In general the penetrating ions
ically using riboflavin phosphate as a catalyst or chem- move through the pores with mobilities commensurate
ically using chemical catalysts. Once the initiation has with those measured in free solution. It is usually diffi-
occurred, the acrylamide crosslinks with itself, with the cult to measure these mobilities because the path length
evolution of heat to produce linear chains that are termi- taken by the ion is unknown, although it is certainly greater
nated when the activated chain ends are neutralized by than the simple linear distance measured macroscopically
collision with other free radicals from the solvent. The along the gel. This means that PAGE cannot yield physico-
product is a stable gel containing only a few percentage chemical data on absolute charges of diffusible ions; only
points (by weight) of polyacrylamide. Gels formed from relative mobilities can be obtained and estimated by ref-
only acrylamide have many undesirable properties (e.g., erence to standards electrophoresed in the same gel.
they are glutinous and stick to glass), and the pore sizes In the case of macroions the relationship between rela-
are illdefined. A more satisfactory gel was formed by in- tive mobilities and charge is complicated by the possibility
cluding a small proportion of the bifunctional acrylamide that selective retardation of the ions occurs as a result of
(Fig. 4b) in the mixture. This forms random cross-links physical impedence by the gel matrix and adsorption onto
between the linear chains during polymerization. The av- the stationary polyacrylamide. Contributions from both
erage size of the pores formed by this mixture is deter- occur with all ions, but for proteins the adsorption is less
mined by the proportion of bisacrylamide (Fig. 4b) to the important than the filtering. At the simplest level this fil-
normal acrylamide in the original solution. The mechan- tering would be described through frictional coefficients
ical stability of the gel decreases with increased propor- [Eq. (6)], which for a fixed pore size increase in proportion
tion of crosslinking, and for some purposes the bisacry- to the cube root of the mass. Thus, the velocity is propor-
lamide is replaced by N,N -diallyltartardiamide. The tional to M −1/3 , and the relative position of macroions on

latter produces a gel that is more restrictive to macroions a gel at the end of an experiment are a function of charge
than bisacrylamide at high proportions of cross-linker, but and mass (that is, proportional to Q/M 1/3 ), which means
in turn it is more manageable. The solvent is held in these that the charge cannot be measured independently of the
pores, and the whole gel can be handled without mechan- mass. Because of this proportional dependence of relative
ical supports, despite the fact that it consists of up to 95% positions on the two dependent parameters, one must be
(by weight) of solvent. fixed before the other can be estimated using relative mo-
Polyacrylamide gel electrophoresis (PAGE) is carried bilities. Thus, if Q were constant for the macroions in a
out using simple equipment. The gels are cast either in mixture, the effective van der Waals radii could be esti-
glass tubes or as slabs supported on nonconducting plates, mated for each from its relative mobility, and if the shape
which can themselves be thermostatted. Because the gels of the ions were constant (that is, all spheres), this would
are generally thin, they have low electrical conductance, so give relative masses M.
relatively high voltages can be applied without excessive These criteria have been used extensively for measuring
heating(say,1000Vacrossa15-cmtube,2-mmdiameter). relative masses of proteins and nucleic acids by including
Along with the developments of PAGE were the pro- in a separate channel a mixture of standard proteins or
duction of a variety of organic ions that would buffer pH nucleic acids of known relative masses; the masses are es-
without producing solutions of high conductivity. High timated in advance using absolute procedures such as that
conductivity arises because of high mobilities of ions, and involving the analytical ultracentrifuge. It is usually not
this is inversely proportional to the van der Waals radius of possible to measure charge this way, because a range of
an ion [Eq. (6); the frictional coefficient is proportional to macroions are not usually available having a fixed mass
this radius]. Organic ions have larger radii than inorganic but carrying a range of charges. It is possible to generate
ions such as phosphates, and so their conductivity is less. such a standard by progressive carbamylation of amino
groups (easily achieved by cautions warming of a protein
with urea solutions) to produce secondary amines that are
B. Molecular Properties Important for
not charged at neutral pH. If the carbamylation is not al-
Polyacrylamide Gel Electrophoresis
lowed to go to completion for all the side chains, this
When ions are transported through the pores of the gel, will produce a mixture of molecules of relatively constant
their relative mobilities can be used to determine their mass (the carbamyl group has a relative mass of 60, so
sizes, provided that they are not excluded from the pores even adding 10 groups to one macroion will not signifi-
of the gel. The radii of the transporting ions are not simple cantly increase the mass). This standard mixture can be
to define because for both the stationary matrix and the used to determine relative charges of the unknowns; then
diffusible ions there are layers of solvent that are trans- if the original absolute charge and amino acid composition