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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
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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 (),

= 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 = f
f is the frictional coefficient, describes frictional resistance to
mobility and depends on a number of factors such as the mass
of the molecule

is the velocity

E is the electric field

q is the net charge on 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% 16%

Dr Gihan Gawish
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 DrpH 8.6, which


Gihan Gawish
is commonly used, all the proteins are
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 the preferred


(greater than a few matrix
hundred bases) *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 overlapping indistinguishable multiple


the components bands smears unresolved
components.

Dr Gihan Gawish
Gel Electrophoresis- Visualization
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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 contain molecules that means they are
at the same passed through the gel approximately the
distance from the with the same speed same size
top
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. The image is recorded with a

Animation computer operated camera

Dr Gihan Gawish