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1. M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2. 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

3. 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,.

4. 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.

5. 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.

6. Description of technique
Instrumentation and Reagents
A schematic diagram of a conventional electrophoresis system
is shown

7. 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.

8. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15.  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.

16. 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)

17.  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 .

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

19. 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.

20. 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.

21. 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.

22. 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

23. 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.

24. 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.

26. 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.

27. 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

28. 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.

29. 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.

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

31. 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.

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

33. 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.,

34. 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.

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

36. 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.

37. 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

38. Albumin>α1 Globulin> α2 Globulin>β
Globulin>Gamma 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

40. 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.

41.  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.

43. 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.
.

44. 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.

45. 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.

46. 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

48. 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)

49. 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.

51. 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).

52. 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.

53. 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.


54. 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.

56. 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.

57. 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.

58. 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.

59. 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.

60. 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.

62. 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.

63. 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 .

64. 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).

65. 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.