9
Electrophoresis
Most biological polymers are electrically charged and will therefore
move in an electric field. The transport of particles through a solvent
by an electric field is called electrophoresis.
A useful way to characterize macromolecules is by their rate ol
movement in an electric field. This property be used to determine
can
protein molecular
net charge or their
weights, distinguish molecules by virtue of their
to
shape, to detect amino acid changes from charged to
uncharged residues or vice versa, and to separate different molecular
species quantitatively. How this is done is described in this chapter.
Theory of Electrophoresis
The detailed theory of
electrophoresis is highly
present incomplete; a simple complicatedinle
is suficient for an description of the electrophoretic princip
purposes.
understanding of how the technique is used 1or most
In many ways,
electrophoresis
force is apPplied and countered
is like sedimentation (Chapte
a
by
viscous
suspended in an insulating medium, is in drag. Ifa particle
an electric with
field, E, thechars
partic.
will move at a constant
velocity, v, determined by the balance
the electrical force, Eq, and
betv
n
the viscous tional
coefficient, that is, drag, fv, in which f is theu
276
Eq = fv (0
, 277
1t is important velocity is proportional to the voltage
to note that the
This
and that the electrical current does not appear in equation (1). Electrophoresis
discussed in some detail later in this chapter.
noint will be
P the velocity per unit field, or
The mobility, u, is defined as
u =
v/E =
q/S (2)
is
mobility depends on the frictional coefficient, which in
turn
Because
the molecules, in
a function of some of the physical parameters of
should give information about the size and shape
principle the value of u
molecule. However, the supporting medium is
normally not an
of the
insulator but an electrolyte consisting
of charged ions, and this intro-
The principal complication is that a charged
duces great complexity.
attracts the ions and hence is sur-
particle suspended in an electrolyte
shields the particle from the applied
rounded by an ion atmosphere that
is partly disrupted both by the
field. However, this ion atmosphere
the medium. To date,
field and by the motion of the particle through
failed to account adequately for these
the theory of electrophoresis has
has not
complications, as well as several others, so that electrophoresis
information about
detailed
turned out to be very useful in supplying
macromolecular structure. It is, however, enormously
useful as both an
of its ability to separate
analytical and a preparative tool because
correct in providing the
different molecules. Although the theory is
thát mobility increases with q,
working rule of electrophoresis-that is,
decreases with f, and is zero for uncharged molecules-in practice,
almost always determined
theoptimal conditions for separation are
empirically.
Types of Electrophoresis
and zone.
There are two types of electrophoresis: moving-boundary
Moving-Boundary Electrophoresis
In moving-boundary electrophoresis, macromolecules are present
of the molecules (actually, the
throughout a solution and the position
from the solvent) as a function of time
Doundary separating the solution This method, which is in
Is determined
by schlieren optics (Chapter 11).
sedimentation, is an analytical
many ways equivalent to boundary
method that has been used primarily for the determination of mobilities
Reference will often be made to preparative and analytical procedures.,
By a preparative
relatively large quantities of pure
procedure is meant a technique designedlaterprovide
to
is
(or nearly pure) material to be used in ex periments. An analytical procedure
mixture and
the number of conmponents in a
used in determining purity, evaluating changes in charge, and so lorth that is,
posSibly the proportions of each, detecting
information to find answers to particular questions.
Oblaining
Electrophoresis
Most biological polymers are electrically charged and will therefore
move in an electric field. The transport of particles through a solvent
by an electric field is called electrophoresis.
A useful way to characterize macromolecules is by their rate ol
movement in an electric field. This property be used to determine
can
protein molecular
net charge or their
weights, distinguish molecules by virtue of their
to
shape, to detect amino acid changes from charged to
uncharged residues or vice versa, and to separate different molecular
species quantitatively. How this is done is described in this chapter.
Theory of Electrophoresis
The detailed theory of
electrophoresis is highly
present incomplete; a simple complicatedinle
is suficient for an description of the electrophoretic princip
purposes.
understanding of how the technique is used 1or most
In many ways,
electrophoresis
force is apPplied and countered
is like sedimentation (Chapte
a
by
viscous
suspended in an insulating medium, is in drag. Ifa particle
an electric with
field, E, thechars
partic.
will move at a constant
velocity, v, determined by the balance
the electrical force, Eq, and
betv
n
the viscous tional
coefficient, that is, drag, fv, in which f is theu
276
Eq = fv (0
, 277
1t is important velocity is proportional to the voltage
to note that the
This
and that the electrical current does not appear in equation (1). Electrophoresis
discussed in some detail later in this chapter.
noint will be
P the velocity per unit field, or
The mobility, u, is defined as
u =
v/E =
q/S (2)
is
mobility depends on the frictional coefficient, which in
turn
Because
the molecules, in
a function of some of the physical parameters of
should give information about the size and shape
principle the value of u
molecule. However, the supporting medium is
normally not an
of the
insulator but an electrolyte consisting
of charged ions, and this intro-
The principal complication is that a charged
duces great complexity.
attracts the ions and hence is sur-
particle suspended in an electrolyte
shields the particle from the applied
rounded by an ion atmosphere that
is partly disrupted both by the
field. However, this ion atmosphere
the medium. To date,
field and by the motion of the particle through
failed to account adequately for these
the theory of electrophoresis has
has not
complications, as well as several others, so that electrophoresis
information about
detailed
turned out to be very useful in supplying
macromolecular structure. It is, however, enormously
useful as both an
of its ability to separate
analytical and a preparative tool because
correct in providing the
different molecules. Although the theory is
thát mobility increases with q,
working rule of electrophoresis-that is,
decreases with f, and is zero for uncharged molecules-in practice,
almost always determined
theoptimal conditions for separation are
empirically.
Types of Electrophoresis
and zone.
There are two types of electrophoresis: moving-boundary
Moving-Boundary Electrophoresis
In moving-boundary electrophoresis, macromolecules are present
of the molecules (actually, the
throughout a solution and the position
from the solvent) as a function of time
Doundary separating the solution This method, which is in
Is determined
by schlieren optics (Chapter 11).
sedimentation, is an analytical
many ways equivalent to boundary
method that has been used primarily for the determination of mobilities
Reference will often be made to preparative and analytical procedures.,
By a preparative
relatively large quantities of pure
procedure is meant a technique designedlaterprovide
to
is
(or nearly pure) material to be used in ex periments. An analytical procedure
mixture and
the number of conmponents in a
used in determining purity, evaluating changes in charge, and so lorth that is,
posSibly the proportions of each, detecting
information to find answers to particular questions.
Oblaining
