ELECTOPHOROSIS

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ELECTROPHORESIS
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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• ELECTROPHORESIS
• Electrophoresis use the migration of Charged
  Solutes or particles in liquid medium under the
  influence of an electric field.
• Electrophoresis is widely used analytical
  technique for the seperation of biological
  molecules such as plasma proteins, lipo
  proteins,immunoglobulins,abnormal hemoglobins,
  genetic variants of transferrin and haptoglobins
  and isoenzymes

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General Principle of electrophoresis This
technique is used for the separation of charged
particles. Biological materials such as amino
acids, peptides, proteins, nucleic acids posses
ionizable groups and hence exist as charged
molecules in solutions, either as cations (vely
charged) or anions(-vely charged) depending upon
the pH of the medium,.
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contd

• Even typical nonpolar substance such as
  carbohydrates can be given charges by
  derivatization, for example as borate or
  phosphates.
• These charged particles like cations move towards
  cathode(-vely charged electrode) and anions
  towards anode (vely charged electrode) in an
  electric field.

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• The difference in Charge Mass ratio (C/M) forms
  the basis for the differential migration of
  particles in an applied electric field. This
  forms the general principle of electrophoresis.
• The Rate of migration of the charged particle
  depends on
• The amount of net charge on the solution
• The size and shape of the solute particles
• The electrical gradient established in the
  solution
• The pH of the solution
• Particles of like charges move in an electric
  field on the basis of their net charge, size, and
  shape.

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• Description of technique
• Instrumentation and Reagents
• A schematic diagram of a conventional
  electrophoresis system is shown

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• Made of either platinum or carbon, the polarity
  of which is determined by the mode of connection
  to the power supply.
• The electrophoresis support
• On which separation takes place may contact the
  buffer directly, or by means of wicks
• Two buffer boxes 1. With baffle plates contain
  the buffer used in the process. Each buffer box
  contains an electrode.
• The entire apparatus is covered
• To minimize evaporation and protect the system
  and is powered by a direct current power supply.

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• Buffers
• The buffer serves as a multifunctional component
  in the electrophoretic process as it,
• Carriers the applied current.
• Establishes the pH at which electrophoresis is
  performed and
• Determines the Electrical charge on the solute.

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As the ionic strength of the buffer increases,
the proportion of current carried by the buffer
will increase and the share of the current
carried by the sample will decrease, thus slowing
down the rate of migration. So always ionic
strength of 0.05M is preferred for the separation
of proteins, or lipoproteins in an electric field.
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Support Media The Support Medium provides the
matrix in which separation takes place.
Various types of support media are used in
electrophoresis and vary from pure buffer
solution in a capillary to insoluble gels (e.g.,
sheet, slabs, or columns of starch, agarose, or
polyacrylamide) or membrances of cellulose
acetate.
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contd

• Gels are cast in a solution of the same buffer to
  be used in the procedure and may be used in a
  horizontal or vertical direction.
• In either case, maximum resolution is achieved if
  the sample is applied in very fine starting zone.
  
• Separation is based on difference in
  charge-to-mass ratio of the proteins and,
  depending on the pore size of the medium.

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Starch Gel and Cellulose Acetate Starch gel was
the first material to be used as a support medium
for electropheresis. Because preparation of a
reproducible starch gel is difficult, this medium
is now rarely used in the clinical laboratory.
Cellulose acetate are seldom used in routine
clinical applications. Currently,agarose and
polyacrylamide gels are the support media of
choice for electrophoresis.
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• Agarose
• Agarose is used in a agarose gel electrophoresis
  (AGE) for the separation of 1.serum, urine, or
  cerebrospinal fluid (CSF) proteins, 2. Hemoglobin
  variants, 3. Isoenzymes, 4.lipoproteins, and 5.
  Other Substances.

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Most routine procedure for AGE are carried out on
commercially produced, prepacked microzone
gels. Operationally, 0.5 to 1.0 of agarose/DL of
buffer provides a gel with suitable strength and
good migration properties for proteins and DNA
fragments in the range of 0.5 to 20.0 kbp
(kilobase pairs)
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• Polyacrylamide
• Polyacrylamide is most useful for mixtures of
  smaller DNA fragments
• The average pore size in a typical 7.5
  polyacrylamide gel is about 5nm(50A).
• With polyacrylamide, proteins are separated, on
  the basis of both charge to mass ratio and
  molecular size, a phenomenon referred to as
  molecular sieving .

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contd

• Because of the potential carcinogenic character
  of acrylamide appropriate caution must be
  exercised when handling this material if gels are
  prepared manually

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Automated Systems Many laboratories are
converting to automated system for
electrophoresis, such as the helena SPIFE 3000 or
the Sebia Hydragel-Hydrasys system. They are
capable of processing of 10 to 100 samples
simultaneously. Beckman Coulter Capillary Zone
Electrophoresis (CZE) system permits
simultaneous processing of seven sample by using
multiple capillaries.
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contd

• Newer microchip based analyzers like the Agilent
  2100 Bioanalyzer significantly miniaturize and
  increase the speed of the process for separating
  proteins, nucleic acids even entire cells.

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GENERAL procedure General Operations performed
in conventional electrophoreis include 1.
Separation, 2.Staining 3.Detection, and 4.
Quantification. Separation To perform an
electrophoretic separation, a hydrated support
material such as precast microzone agarose or
polyacrylamide, gel is blotted to remove excess
buffer and then placed into the electrophoresis
chamber.
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contd

• Care should be taken that the gel has neither
  excess liquid nor bubbles on it.
• Next the sample is added to the support and it
  is placed in contact with buffer previously
  added to the electrode Chambers.
• Electrophoresis is conducted for a determined
  length of time under conditions of either
  constant voltage or constant current

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Staining When electrophoresis is completed, the
support is removed from the electrophoresis cell
and rapidly dried or placed in a fixative to
prevent diffusion of sample components. It is
then stained to visualize the individual protein
zone. After washing out excess dye, the support
is dried.
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contd

• Stains used to visualize the separated protein
  fractions are differ according to type of
  application.
• Most commercial methods for serum protein
  electrophoresis use Amido Black B or members of
  the Coomassie Brilliant Blue series of dyes for
  staining.
• Silver nitrate or silver diamine has been used
  to stain proteins and polypeptides.

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Detection and Quantification Once electrophoretic
separation and staining are complete, it is
possible to quantify the individual zones either
as a percentage of the total or as absolute
concentration by direct densitometry, If the
total protein concentration is known.
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contd

• In the densitometer, the gel (or other medium) is
  moved past a measuring optical system and the
  absorbance of each fraction is displayed on a
  recorder chart or an electronic display

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Modern DNA analysis techniques, which may produce
several dozen bands of different length DNA
fragments require a new type of densitometer
referred to as a flat bed scanner or digital
image analyzer.
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contd

• In addition to scanning by densitometry,
  electrophoresis gels are now being analyzed by
  mass spectrometers to determine the molecular
  weights of proteins and their cleavage products,
  and for peptide sequencing.

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Types of Electrophoresis There are mainly two
types of Electrophoresis, 1. Vertical
Electrophoresis 2. Horizontal Electrophoresis Diff
erent types of support media are used in
Horizontal Electrophoresis Ex Filter Paper ,
cellulose acetate agar gel, agarose gel, starch
gel, etc.,
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contd

• The Vertical Electrophoresismainly use
  polyacrylamide gel,
• Depending upon the nature of supporting medium.
• Agar gel Electrophoresis AGE)-where agar gel is
  used as supporting medium.
• Polyacrylamide gel Electrophoresis(PAGE).
  Acrylamide and methylene bisacry lamide forms a
  polymer in this case.
• Cellulose acetate electrophoresis (Paper Strip
  Electrophoresis)
• Where cellulose acetate paper serves as the
  supporting medium.

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• 2. Depending upon the mode of the technique
• Slide gel Electrophoresis
• Tube gel Electrophoresis
• Disc Electrophoresis
• Low and high voltage Electrophoresis

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Zone Electrophoresis Zone Electrophoresis
techniques produce zone of proteins, which are
heterogeneous and physically separated from one
another as shown . They are classified
according to the type and structure of the
support material used and are commonly referred
to as AGE cellulose acetate electrophoresis (CAE)
polyacrylamide gel Electrophoresis (PAGE)etc.,
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contd

• In 1934, consden introduced a simpler Zone
  electrophoresis technique using filter paper as a
  supporting medium .
• It uses an inert supporting like paper or gel.
  It is simple, routinely used and gives better
  resolution than moving boundary type.
• A simple and modified method of moving boundary
  electrophoresis is the zone electrophoresis.

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• Various Types of Zone electrophoresis
• 1. Paper Electrophoresis
• 2. Gel Electrophoresis
• 3. Isoelectric Focusing
• 4. Cellulose acetate
• 5. Immunoelectrophoresis

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Free or Moving Boundary Electrophoresis The
Original moving boundary Electrohoresis system
was devised by arne Tiselius in 1937. Arne
Tiselius was awarded nobel prize in 1948 Tiselius
1937 introduced this technique This consists of
a quartz U-shaped tube.
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contd

• Protein in buffer (pH-8-6) is taken in it and is
  layered with protein free buffer in both limbs of
  the tube.
• Two electrodes are fitted in the two limbs of
  cell and joined to the battery.
• When electric current is passed through the
  U-tube, the protein negatively charged migrate
  towards anode and their rate of migration is

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Albumingta1 Globulingt a2 Globulingtß
GlobulingtGamma Globulin When current has flown
through for 16 hours front edge of albumin has
travelled considerably ahead of other proteins in
the order given above. The position of the
boundary for each protein can be determined by
observing the change in refractive index. The
refraction can be photographed in the form of so
called electrophoretic pattern
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contd

• Separation only occurs at the boundaries.
• The bulk of the protein remaining homgenous.
• It is called moving or free boundary because
  proteins move freely in solution.
• This apparatus is complicated and costly and so
  was comparatively little used.

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• Paper Electrophoresis
• The technique of paper electrophoresis is simple
  and inexpensive and requires only micro
  quantities of plasma for separation.
• The electrophoresis apparatus in its simplest
  form consists of two troughs to contain buffer
  solution, through which electric current is
  passed.

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The plasma under investigation is mixed with
bromophenol blue, a blue coloured stain and
spotted at the centre of a strip of special
filter paper, saturated with an alkaline buffer
solution. Barbitone Buffer of pH 8.6 is used.
The centre of the filter paper is supported
vertically over a glass rod. The ends of the
paper dip in buffer solution contained in two
troughs placed on either side of the glass rod.
The entire system is covered with an airtight
transparent lid. .
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An electric current of proper amperage and
voltage is passed Across the paper, when the
charged protein fractions bearing different
charges migrate at different rates. The moving
boundary of the migrating proteins, stained blue
can be seen moving towards anode.
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contd

• After a run of about five to six hours, the
  current is stopped and the separated fractions of
  plasma protein which lie in succession behind the
  moving boundary, are fixed in their places by
  heating the paper in an oven at 110 c for half
  an hour.
• The paper is then stained with a solution
  containing bromophenol blue.

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The different fractions appear as blue coloured
bands across the filter paper starting from the
moving boundary backwards. If a quantitative
estimation is required for each fraction, the
bands may be carefully cut and eluted, or the
bands may be scanned optically in a
densitometer. In human plasma five different
bands can be identified on paper electrophoresis
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GEL ELECTROPHORESIS This technique
involves the separation of molecules based on
their size, in addition to the electrical charge.
The movement of large olecules is slow in gel
electrophoresis. Serum proteins can be
separated to about 15 bands, instead of 5 bands
on paper electrophoresis. The gels commonly
used in gel electrophoresis are agarose and
Polyacrylamide, sodium dodecyl sulfate (SDS)
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AGAR GEL ELECTROPHORESIS Slide Preparation
About 1.4 ml of Warm (60 c) agar solution (100
mg / 10 ml of Barbitone buffer) is delivered on a
slide uniformly at room temperature and allowed
to solidify. Chamber saturation Twenty minutes
before starting the experiment, both the buffer
tanks of the electrophoretic chamber are filled
with equal volume of 0.05 M barbitone uffer
(pH8.6) and kept closed to saturate the chamber
With solvent vapors.
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Sample Application Small filter paper strip
(Whatman No 1, 1 X 5 mm ) Soaked in the Serum
sample is kept on the slide perpendicular to the
length of the slide Close towards the cathode and
the chamber is closed. Slide Projection and
Wick Connection The slide prepared is kept in
the chamber and connected to buffer By means of
filter paper strips (Wicks).
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Application of Electric Field The
electrophoretic chamber is connected to a power
pack (an Equipment used to alter or adjust the
required current or voltage values). The power
pack is switched on and current is adjust so that
4 m Amp current Flows through each slide. The
process has to be carried out for about 90-120
minutes.
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contd

• Fixing of Proteins
• The slides are kept immersed in the absolute
  alcohol for about 20 Minutes to prevent the
  diffusion of separated proteins.
• Staining
• The Slides are dried and kept immersed in the
  Amidoschwartz 10B dye For about 5 minutes.

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Destaining The Stained slides are washed with 3
acetic acid to clear the background and to see
the protein bands clearly. The dried slides can
be preserved for a long time. Since the
electrophoretic pattern of serum proteins in
certain diseases vary markedly from a normal
pattern it is of great diagnostic significance is
several conditions like nephrosis, liver
disease, multiple myeloma, gamma globulinemia and
others.
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Polyacrylamide gel electrophoresis (PAGE ) It
is useful when high resolution is required.
Addition of a detergent sodium dodecyl sulphate
(SDS) makes it the convenient technique for
separation of proteins and determination of their
molecular weight, and also for separation of
isoenzymes. Polyacrylamide is employed for the
determination of molecular weights of proteins in
a popularly known electrophesis technique known
as SDS-PAGE.
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ISOELECTRIC FOCUSSING It is the improved type
of electrophoresis. It separates proteins
Electrophoretically on the basis of their
relative content of positively and negatively
charged groups and isoelectric pH values. It is
useful for separation of isoenzymes and some
proteins of biological Importance.
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This technique is primarily based on the
immobilization of the molecules at isoelectric pH
during electrophoresis. Stable pH gradients are
set up (usually in a gel) covering the pH range
to include the isoelectric points of the
components in a mixture. As the electrophores is
occurs, the molecules (say proteins ) migrate to
positions corresponding to their isoelectic
points , get immobilized and form sharp
stationary bonds.
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The gel blocks can be stained and identified. By
isoelectic focussing, serum Proteins can be
separated to as many as 40 bands. Isoelectric
focussing Can be conveniently used for the
purification of proteins. CELLULOSE ACETATE
ELECTROPHORESIS The strips of cellulose
acetate are used as supporting medium,Particularly
in separation of variants of hemoglobin.
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IMMUNOELECTROPHORESIS Here antigenic proteins
are separated by combining their electrophoretic
migration with their precipitation by specific
antibodies. This technique involues combination
of the principles of Electrophoresis and
immunological reactions. Immunoelectrophoresis
is useful for the analysis of complex mixtures of
antigens and Antibodies.
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The complex proteins of biological samples (say
human serum) Are subjected to electrophoresis.
The antibody (antihuman immune Serum from rabbit
or horse ) is then applied in a trough parallel
to the Electrophoretic separation. The
antibodies diffuse and, when they come In contact
with antigens, Precipitation occurs, resulting in
the formation Of precipitin bands which can be
identified.
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APPLICATION OF ELECTROPHORESIS Clinical diagnosis
a. Separation of serum proteins Different
patterns are seen with different diseases. For
ex, in nephrotic syndrome, albumin and gamma
Globulin bands are decreased while alpha-2 band
is increased .
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In multiple myeloma, there will be a M band
(paraprotein) in Gamma region or gamma-beta inter
region. b. Useful in separation of isoenzymes
(LDH, ALP,CPK etc) in differential Diagnosis,
hemoglobin variant (hemoglobino-pathies),
lipoproteins (hyperlipoproteinemias).
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2. SDS PAGE used for finding molecular weight
of proteins and their Purification. 3.
Separation and analysis of nucleotides and
nucleic acids, DNA finger printing. 4.
Hemoglobin separation 5. Lipoprotein separation
and identification 6. Determination of
molecular weight of proteins.
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