Download 3-1 Electrophoresis and its types (Moving Boundary Electrophoresis

1
If a mixture of electrically charged biomolecules
is placed in an electric field of field strength E,
they will freely move towards the electrode of
opposite charge.
Dr Gihan Gawish
Theory of Electrophoresis
2

Electrophoretic separations are based upon the fact that the
electrical force (F) on a charged particle (ion) in an electrical
field (E) is proportional to the charge of the particle (q),
F = qE

The migration of the charged particle in the electric field, called
the electrophoretic mobility (μ),
μ = v/E = q/f
Dr Gihan Gawish
Theory of Electrophoresis
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
Move at quite different depending on
 physical characteristics of the molecule
 experimental system used.
Dr Gihan Gawish
Theory of Electrophoresis
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Eq = ‫ע‬f

f

‫ ע‬is the velocity

E is the electric field

q is the net charge on molecule
is the frictional coefficient, describes frictional resistance to
mobility and depends on a number of factors such as the mass
of the molecule
Dr Gihan Gawish
1- Moving Boundary Electrophoresis
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
Electrophoresis in a free solution.

the separation of colloids through electrophoresis

the motion of charged particles through a
stationary liquid under the influence of an electric
field.
Developed by Arne Tiselius in 1937. Tiselius was awarded the 1948
Nobel Prize in chemistry for his work
Dr Gihan Gawish
Tiselius Electrophoresis
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Moving boundary electrophoresis is electrophoresis in a free solution.

The apparatus includes a U-shaped cell filled with buffer solution
and electrodes immersed at its ends.

On applying voltage, the compounds will migrate to the anode or
cathode depending on their charges.
The sample applied could be any mixture of charged components like a
serum
Dr Gihan Gawish
Tiselius Electrophoresis
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
Moving boundary electrophoresis is electrophoresis in a free solution.
The principle is the motion of
charged particles through a
stationary liquid under the
influence of an electric field.
59% 8% 5%12%
Dr Gihan Gawish
16%
2- Zone Electrophoresis
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
separation cellulose acetate electrophoresis.

A drop of sample is applied in a band to a thin sheet of supporting
material, like paper, that has been soaked in a slightly-alkaline salt
solution .

Plasma protein example: At pH 8.6, which is commonly used, all
the proteins are negatively charged, but some more strongly than
others.
Dr Gihan Gawish
Zone Electrophoresis
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
A direct current can flow through the paper because of
the conductivity of the buffer

The serum proteins move toward the positive
electrode .

The stronger the negative charge on a protein, the
faster it migrates .
Dr Gihan Gawish
Zone Electrophoresis
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
After 20 min, the current is
turned off

the proteins stained to make
them visible

The separated proteins appear
as distinct bands .

serum albumin moves closest
to the positive electrode, why?
Dr Gihan Gawish
3- Gel Electrophoresis
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

The term "gel" in this instance refers to the matrix
used to contain, then separate the target
molecules.
In most cases the gel is a cross linked polymer
whose composition and porosity is chosen based
on:
 the specific weight
 composition of the target to be analyzed.
Dr Gihan Gawish
Gel Electrophoresis
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larger nucleic acids
(greater than a few
hundred bases)

the preferred
matrix
*agarose.
*Acrylamide, in contrast to *Polyacrylamide, is a
neurotoxin and must be handled using appropriate
safety precautions to avoid poisoning.
Dr Gihan Gawish
Gel Electrophoresis- Visualization
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Ethedium bromide
Silver
stained
Photograph
Autoradiogram
Dr Gihan Gawish
Gel Electrophoresis- Visualization
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
If several mixtures have initially been injected next to
each other, they will run parallel in individual lanes.
Incomplete separation of
the components
overlapping
bands
indistinguishable
smears
multiple
unresolved
components.
Dr Gihan Gawish
Gel Electrophoresis- Visualization
15

The bands observed (unknown molecular weight) can be
compared to those of the known (Molecular weight size
markers) in order to determine their size.

Molecular weight size markers contain a mixture of molecules of
known sizes.

marker run on one lane in the gel parallel to the unknown
samples
Bands in different
lanes that end up
at the same
distance from the
top
contain molecules that
passed through the gel
with the same speed
means they are
approximately the
same size
Dr Gihan Gawish
Applications of Gel Electrophoresis
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Forensics,

The results can be analyzed
Molecular biology,
quantitatively by visualizing the
Genetics
gel with UV light and a gel
 Microbiology
imaging device.
 Biochemistry.
Animation

The image is recorded with a
computer operated camera
Dr Gihan Gawish