|
|
|
|
Gel
Electrophoresis -
Electrophoresis
is
the
technique
of
separation
of
charged
molecules
under
the
influence
of
an
electrical
field
so
that
they
migrate
in
the
direction
of
electrode
bearing
the
opposite
charge,
viz,
cationic
(positively
charged)
molecules
move
toward
cathode
(-ve
electrode)
and
anionic
(negatively
charged)
molecules
travel
towards
anode
(+ve
electrode).
The
molecules
to
be
separated
are
maintained
in
aqueous
phase.
The
speed
of
migration
(electrophoretic
mobility)
of
a
molecule
depends
on
its
charge
and
molecular
mass.
Charge
of
a
molecule
is
influenced
by
the
following:
(1)
the
type,
concentration
and
pH
of
buffer,
(2)
the
temperature,
(3)
strength
of
the
electrical
field,
and
(4)
the
nature
of
the
support
material
(matrix)
used
for
electrophoresis.
|
Types
of
Electrophoresis
There
are
basically
three
different
electrophoretic
methods
as
follows:
(1)
electrophoresis
(sometimes
called
zone
electrophoresis),
(2)
isotachophoresis
and
(3)
isoelectric
focussing.
A
huge
variety
of
electrophoretic
methods
have
been
devised
to
achieve
specific
objectives;
for
greater
details
and
procedural
directives/variations,
the
reader
is
advised
to
consult
one
of
the
following
books.
1.
Andrews,
A.T.
1986.
Electrophoresis,
Theory,
Techniques,
and
Biochemical
and
Clinical
Applications.
Clarendon
Press,
Oxford.
2.
Chrambach,
A.
1985.
The
Practice
of
Quantitative
Gel
Electrophoresis.
VCH,
Weinheim.
3.
Mosher,
R.A.,
Saville,
D.A.
and
Thorman,
W.
1992.
The
Dynamics
of
Electrophoresis.
VCH,
Weinheim.
4.
Westermeier,
R.
1993.
Electrophoresis
in
Practice.
VCH,
Weinheim.
|
Electrophoresis
can
be
carried
out
in
free
solutions,
e.g.,
capillary
electrophoresis,
or
in
a
stabilizing
support
material
like
thin
layer
plates,
films
and
gels.
A
brief
description
of
gel
electrophoresis
is
given
below.
Gel
electrophoresis Gel
matrices
should
have
adjustable
and
regular
pore
size,
should
be
chemically
inert
and
should
not
exhibit
electroosmosis.
Electroosmosis
is
the
phenomenon
of
migration
of
water
toward
an
electrode
as
a
result
of
the
supporting
medium
and/or
the
surface
of
the
separation
equipment,
e.g.,
of
capillaries,
also
carrying
charge.
The
gel
can
be
as
vertical
rods,
as
plates
or
horizontal
slabs.
The
following
types
of
gels
are
commonly
used.
1.
Agarose
Gels These
gels
have
large
pores,
and
are
used
for
analysis
of
molecules
of
over
10
nm
diameter.
Agarose
is
a
polysaccharide
obtained
from
red
seaweed.
When
agaropectin
is
removed,
agarose
gells
with
melting
points
from
35°C
to
95°C
and
varying
degrees
of
electroosmosis
are
obtained.
Agarose
dissolves
in
hot
water.
|
|
When
this
solution
is
cooled,
double
helices
form
and
become
arranged
laterally
the
produce
thick
filaments;
these
filaments
become
cross
linked
to
form
the
gel.
Pore
size
depends
on
agarose
concentration
(w/v):
in
general,
a
1
%
(w/v)
gell
will
have
a
pore
size
of
150
nm,
while
a
0.16%
gel
has
pore
size
of
500
-nm.
Gels
having
0.7
to
1
%
agarose
have
large
pore
size.
Such
horizontal
I
agarose
gels
are
used
for
the
separation
of
high
molecular
weight
proteins,
e.g.,
serum
proteins,
and
enzymes,
e.g.,
isoenzymes
of
diagnostic
importance,
and
of
large
(few
to
several
kb)
fragments
of
DNA.
Proteins
separated
in
agarose
gels
can
be
subjected
to
immunofixation,
immunoprinting
and
immunoblotting.
Agrarose
gels
are
also
used
for
Immunoelectrophoresis
and
affinity
electrophoresis.
|
|
|
Agarose
gels
above
1
%
concentration
are
cloudy
and
exhibit
high
electro
osmotic
flow.
These
gels
are,
therefore
used
for
the
separation
of
very
high
molecular
weight
proteins
or
protein
aggregates.
However,
they
are
the
standard
medium
for
separation,
identification
RFL-analysis,
and
purification
of
DNA
and
RNA
fragments;
for
these
applications,
horizontal
submarine
agarose
gels
are
used
to
prevent
the
gel
from
drying
out.
2.
Polyacrylamide
Gels These
gels
are
obtained
by
copolymerization
of
acrylamide
(CH2=CH-CONH2)
monomers
with
a
cross
linking
reagent
(usually,
N,
N'-methylenebisacrylamide,
bisacarylamide
in
short).
Polyacrylamide
gels
are
transparent,
chemically
inert
and,
particularly,
mechanically
stable,
and
they
exhibit
very
little
electroosmosis.
|
The
temperature
during
polymerization
should
be
maintained
above
20°C
in
order
to
prevent
incomplete
polymerization.
In
addition,
polymerization
should
take
place
under
an
inert
atmosphere
since
oxygen
can
act
as
a
free
radical
trap.
Oxygen
absorption
is
minimized
by
casting
the
gels
in
vertical
chambers,
e.g.,
molds
formed
by
two
glass
plates
sealed
together
around
the
edges
in
the
case
of
flat
gels.
The
monomers
are
toxic;
therefore
they
should
be
handled
with
the
utmost
care.
Horizontal
polyacrylamide
gels
polymerized
on
ultra
thin
films
are
used
to
separate
low
molecular
weight
compounds,
e.g.,
dyes
with
molecular
weights
of
500
Daltons.
Polyacrylamide
gels
are
also
used
for
analysis
of
nucleic
acids,
e.g.,
DNA
sequencing,
for
viroid
tests
(detects
presence
and
also
the
type
of
viroids)
and
to
detect
mutations,
as
well
as
for
analysis
of
proteins.
These
applications
are
briefly
described
below.
The
pore
size
of
the
gel
depends
on
the
following
two
factors:
(1)
the
total
concentration
of
the
acrylamide
and
bisacrylamide
monomers
in
the
solution,
and
(2)
the
proportion
(in
per
cent)
of
bisacrylamide
(C)
in
the
total
monomer
concentration.
The
values
of
T
and
C
are
given
by
the
following
formulae.
When
T
is
increased,
while
C
is
kept
constant,
the
pore
size
of
gel
decreases.
But
when
C
is
increased,
while
T
remains
constant,
the
pore
size
decrease
till
C
equals
4%;
beyond
this
value
of
C,
the
pore
size
again
increases.
The
pore
size
of
polyacrylamide
gels,
therefore,
can
be
exactly
and
predictably
controlled
by
controlling
the
values
of
T
and
C.
It
may
be
pointed
out
that
gels
with
C >5%
are
brittle
and
relatively
hydrophobic;
such
gels
are
used
only
in
special
cases.
| |
