Principle,instrumentation,working mechanism,advantages,limitation &application of MovingBoundary,paper,Immuno,2D,IEF, & Gel(AGE & PAGE) electrophoresis

1. Electrophoresis
Dr Sumitha J,Associate Professor,
JBAS COLLEGE FOR WOMEN,Chennai-18

2. Electrophoresis
● Electrophoresis is a technique used to separate molecules
based on their charge, size, and shape. It is a powerful tool
for analyzing biomolecules, such as DNA, RNA, and
proteins.

6. Principle of Electrophoresis
● The principle of electrophoresis is based on the fact that charged particles
will migrate in an electric field. The direction of migration depends on the
charge of the particle: negatively charged particles will migrate towards
the positive electrode (anode), while positively charged particles will
migrate towards the negative electrode (cathode).
● The speed of migration of a particle is determined by its charge, size, and
shape. Larger particles will migrate more slowly than smaller particles,
and particles with a higher charge will migrate more quickly than particles
with a lower charge

7. Moving boundary electrophoresis

8. MBE
Moving-boundary electrophoresis is a technique used to
separate charged particles based on their net charge. It
is a classic method that was first developed by Arne
Tiselius in the 1930s. Moving-boundary electrophoresis
is based on the principle of electrophoresis, which is the
movement of charged particles in an electric field.

9. Basic principles
Charged particles migrate towards the oppositely charged
electrode at a speed that is proportional to their net charge.
The net charge of a particle is determined by its chemical
composition.
The isoelectric point is the pH value at which a particle has
no net charge.

10. Working mechanism
● A sample of charged particles is placed in a solution.
● An electric field is applied to the solution.
● The charged particles migrate towards the oppositely
charged electrode at a speed that is proportional to their net
charge.
● The particles will eventually reach a point where their net
charge is zero, and they will stop migrating. This point is
called the isoelectric point.

11. Instrumentation
● A Tiselius cell
● A power supply
● A conductivity detector
The Tiselius cell is a U-shaped container that is filled with a
buffer solution. The buffer solution maintains a constant pH
throughout the cell. The power supply provides an electric field
across the cell. The conductivity detector measures the
conductivity of the solution in the cell.

14. Advantages
● High resolution
● Ability to separate small molecules
● Ability to measure the isoelectric point of proteins
● .

15. Limitations
● Complex instrumentation
● Time-consuming
● Not suitable for large molecules

16. Applications
 ● Separation of proteins
 ● Separation of amino acids
 ● Determination of the isoelectric point of proteins

17. Paper electrophoresis

18. Basic principle
Paper electrophoresis is a separation technique that uses an electric field to
separate charged molecules. The molecules are placed on a strip of filter
paper that is soaked in a buffer solution. The buffer solution helps to
maintain a constant pH throughout the paper, which is important because
the charge of a molecule can vary depending on the pH of the solution. When
an electric field is applied, the molecules migrate towards the oppositely
charged electrode. The speed at which a molecule migrates depends on its
charge and size. Larger molecules migrate more slowly than smaller
molecules.

19. Instrumentation
The basic instrumentation for paper electrophoresis consists of
a power supply
a buffer tank
a paper wick
a electrophoresis chamber
Sample applicator
Detector

20. ● Power supply: The power supply provides the electric field that is used to separate
the molecules. The voltage of the power supply is typically adjusted to be between
10 and 20 volts per centimeter.
● Buffer tank: The buffer tank contains the buffer solution that is used to soak the
paper. The buffer solution helps to maintain a constant pH throughout the paper,
which is important because the charge of a molecule can vary depending on the
pH of the solution.
● Paper wick: The paper wick is used to transport the buffer solution from the buffer
tank to the paper. The paper wick is made of a material that is able to transport
the buffer solution quickly and evenly.

21. ● Electrophoresis chamber: The electrophoresis chamber is the container that holds
the paper and the buffer solution. The electrophoresis chamber is typically a
sealed container that prevents the buffer solution from evaporating.
● Sample applicator: The sample applicator is used to apply the sample to the
paper. The sample applicator is typically a small pipette or syringe that is used to
deposit a small amount of the sample onto the paper.
● Detector: The detector is used to detect the separated molecules. The detector can
be a UV lamp or a laser scanner. The UV lamp or laser scanner is used to visualize
the separated molecules on the paper.

22. Working mechanism
The working mechanism of paper electrophoresis is as follows:
● The filter paper is soaked in the buffer solution.
● The sample is applied to the paper in a small spot.
● The power supply is turned on, which creates an electric field.
● The molecules in the sample migrate towards the oppositely charged electrode.
● The molecules are separated according to their charge and size.
● The migration of the molecules is stopped by turning off the power supply.

25. Advantages
The advantages of paper electrophoresis include:
● It is a simple and inexpensive technique.
● It is relatively easy to perform.
● It can be used to separate a wide variety of
molecules.

26. Limitations
The limitations of paper electrophoresis include:
● The resolution of the separation is not as good as other
methods, such as gel electrophoresis.
● The technique is not as sensitive as other methods.
● The technique is not as versatile as other methods.

27. Applications
 The analysis of proteins
 The analysis of amino acids
 The analysis of nucleic acids
 The analysis of enzymes
 The analysis of food colors

28. Gel electrophoresis

29. Basic principle
Gel electrophoresis is a separation technique that uses an electric field
to separate charged molecules. The molecules are placed in a gel that
has pores of a specific size. The smaller the pores, the smaller the
molecules that can pass through them. When an electric field is
applied, the molecules migrate towards the oppositely charged
electrode. The speed at which a molecule migrates depends on its
charge and size. Larger molecules migrate more slowly than smaller
molecules.

30. Instrumentation
The basic instrumentation for gel electrophoresis consists of a power
supply, a gel tank, a comb, and a electrophoresis chamber.
The power supply provides the electric field that is used to separate
the molecules.
The gel tank contains the gel that is used to separate the molecules.
The comb is used to create wells in the gel where the sample can be
applied.
The electrophoresis chamber is the container that holds the gel and
the buffer solution.

31. Working mechanism
The working mechanism of gel electrophoresis is as follows:
● The gel is prepared by mixing a polymer, such as agarose or polyacrylamide, with a
buffer solution.
● The comb is inserted into the gel to create wells.
● The sample is applied to the wells.
● The power supply is turned on, which creates an electric field.
● The molecules in the sample migrate towards the oppositely charged electrode.
● The migration of the molecules is stopped by turning off the power supply.

32. Advantages
The advantages of gel electrophoresis include:
It is a very versatile technique that can be used to separate a
wide variety of molecules, including DNA, RNA, and proteins.
It is a very sensitive technique that can be used to detect very
small amounts of molecules.
It is a very reproducible technique that can be used to produce
consistent results.

33. Limitations
The limitations of gel electrophoresis include:
It can be a time-consuming technique.
It can be a destructive technique, meaning that the molecules
that are being separated are destroyed in the process.
It can be a difficult technique to master.

34. Applications
 The analysis of DNA
 The analysis of RNA
 The analysis of proteins
 The analysis of enzymes
 The analysis of food contaminants

35. Agarose Gel electrophoresis

36. AGE
Agarose gel electrophoresis is a technique that uses agarose, a
polysaccharide extracted from seaweed, to separate DNA
fragments. The agarose gel has large pores, which allows large
DNA fragments to pass through. This makes agarose gel
electrophoresis a good choice for separating large DNA
fragments, such as those produced by PCR.

37. PAGE
Polyacrylamide gel electrophoresis is a technique that uses
polyacrylamide, a synthetic polymer, to separate DNA fragments
and proteins. The polyacrylamide gel has small pores, which
allows only small DNA fragments and proteins to pass through.
This makes polyacrylamide gel electrophoresis a good choice for
separating small DNA fragments and proteins.

40. Basic principle
Agarose gel electrophoresis is a technique that uses an electric field to
separate DNA fragments. The DNA fragments are placed in a gel made
of agarose, a polysaccharide extracted from seaweed. The agarose gel
has pores of a specific size. The smaller the pores, the smaller the DNA
fragments that can pass through them. When an electric field is
applied, the DNA fragments migrate towards the oppositely charged
electrode. The speed at which a DNA fragment migrates depends on its
size and charge. Larger DNA fragments migrate more slowly than
smaller DNA fragments.

41. Instrumentation
The basic instrumentation for agarose gel electrophoresis
consists of a power supply, a gel tank, a comb, and a
electrophoresis chamber. The power supply provides the
electric field that is used to separate the DNA fragments. The gel
tank contains the gel that is used to separate the DNA fragments.
The comb is used to create wells in the gel where the sample can
be applied. The electrophoresis chamber is the container that
holds the gel and the buffer solution.

43. Working mechanism
The working mechanism of agarose gel electrophoresis is as follows:
The gel is prepared by mixing agarose powder with a buffer solution.
The comb is inserted into the gel to create wells.
The sample is applied to the wells.
The power supply is turned on, which creates an electric field.
The DNA fragments in the sample migrate towards the oppositely charged electrode.
The migration of the DNA fragments is stopped by turning off the power supply..

44. Advantages
The advantages of agarose gel electrophoresis include:
It is a relatively simple and easy to perform technique.
It is a versatile technique that can be used to separate a wide
range of DNA fragments.
It is a sensitive technique that can be used to detect very small
amounts of DNA.
It is a reproducible technique that can be used to produce
consistent results.

45. Limitations
The limitations of agarose gel electrophoresis include:
It can be a time-consuming technique.
It can be a destructive technique, meaning that the DNA
fragments that are being separated are destroyed in the process.
The resolution of agarose gel electrophoresis is not as good as
other methods, such as polyacrylamide gel electrophoresis.

46. Applications
 The analysis of DNA fragments
 The identification of DNA mutations
 The detection of DNA contamination
 The study of DNA structure and function

47. Polyacrylamide Gel electrophoresis

48. Basic principle
● Polyacrylamide gel electrophoresis (PAGE) is a technique that uses an electric field
to separate proteins. The proteins are placed in a gel made of polyacrylamide, a
synthetic polymer. The polyacrylamide gel has pores of a specific size. The smaller
the pores, the smaller the proteins that can pass through them. When an electric
field is applied, the proteins migrate towards the oppositely charged electrode.
The speed at which a protein migrates depends on its size, charge, and shape.
Larger proteins migrate more slowly than smaller proteins.

49. Basic principle
● Polyacrylamide gels are chemically cross-linked gels formed by the polymerization
of acrylamide with a cross-linking agent, usually N,N’-methylenebisacrylamide.
● The reaction is a free radical polymerization, usually carried out with ammonium
persulfate as the initiator and N,N,N’,N’-tetramethylethylendiamine (TEMED) as
the catalyst.

50. Instrumentation
The basic instrumentation for polyacrylamide gel
electrophoresis consists of a power supply, a gel tank, a comb,
and a electrophoresis chamber. The power supply provides the
electric field that is used to separate the proteins. The gel tank
contains the gel that is used to separate the proteins. The comb
is used to create wells in the gel where the sample can be
applied. The electrophoresis chamber is the container that holds
the gel and the buffer solution.

51. Working mechanism
The working mechanism of polyacrylamide gel electrophoresis is as follows:
The gel is prepared by mixing acrylamide and bis-acrylamide monomers with a buffer
solution.
The comb is inserted into the gel to create wells.
The sample is applied to the wells.
The power supply is turned on, which creates an electric field.
The proteins in the sample migrate towards the oppositely charged electrode.
The migration of the proteins is stopped by turning off the power supply.

52. We use resolving and stacking gels in PAGE for two
reasons:
● To improve resolution: The resolving gel has a smaller pore size than the stacking
gel, which means that the proteins in the sample will migrate more slowly through
the resolving gel. This helps to improve the resolution of the separation, meaning
that the proteins can be more easily distinguished from each other.
● To concentrate the proteins: The stacking gel has a higher concentration of
acrylamide than the resolving gel, which means that the proteins in the sample
will migrate more quickly through the stacking gel. This helps to concentrate the
proteins at the top of the resolving gel, which also improves the resolution of the
separation.

53. ● Without the stacking gel, the proteins would migrate through the resolving gel at
different speeds, depending on their size and charge. This would make it difficult
to resolve the proteins from each other. The stacking gel helps to ensure that all of
the proteins in the sample migrate through the resolving gel at the same speed,
which improves the resolution of the separation.

58. Advantages
The advantages of polyacrylamide gel electrophoresis include:
It is a very versatile technique that can be used to separate a wide
range of proteins.
It is a very sensitive technique that can be used to detect very small
amounts of proteins.
It is a very reproducible technique that can be used to produce
consistent results.
The resolution of polyacrylamide gel electrophoresis is much higher
than agarose gel electrophoresis.

59. Limitations
The limitations of polyacrylamide gel electrophoresis include:
It can be a more time-consuming and difficult technique to
perform than agarose gel electrophoresis.
It can be a more toxic technique than agarose gel
electrophoresis.
The gel is more fragile than agarose gel, so it is more difficult to
handle.

60. Applications
 The analysis of protein fragments
 The identification of protein mutations
 The detection of protein contamination
 The study of protein structure and function

61. Immuno Electrophoresis

62. Basic principle
● Immunoelectrophoresis (IEP) is a laboratory technique that
combines electrophoresis and immunodiffusion to separate and
identify proteins based on their electrical charge and reactivity
with antibodies.
● In IEP, a sample of proteins is first separated by
electrophoresis in a gel. The separated proteins are then
reacted with antibodies that are specific for those proteins. The
antibodies and antigens will form a precipitate at the point
where they meet, which can be visualized by staining the gel.

63. Basic principle
Immunoelectrophoresis combines principles of
electrophoresis and immunodiffusion. It involves the
electrophoretic separation of proteins in a gel matrix followed
by the immunodiffusion of specific antibodies against the
proteins of interest. When the proteins are separated, they
form distinct bands or precipitin arcs, and by adding specific
antibodies, these bands can be identified and characterized.

65. Instrumentation
The instrumentation required for IEP includes:
• An electrophoresis chamber
• An electric power supply
• A gel electrophoresis kit
• Antibodies specific for the proteins in the sample
• A staining solution

66. Working mechanism
a. Electrophoresis: A mixture of proteins is loaded onto a gel and subjected to an
electric field. Due to their charge-to-mass ratio, the proteins move through the gel
at different rates and get separated based on their size and charge.
b. Immunodiffusion: After electrophoresis, wells or troughs are cut perpendicular
to the direction of protein migration in the gel. Antibodies specific to the protein of
interest are added to these wells, and over time, they diffuse through the gel and
form immune complexes with the corresponding proteins, creating precipitin arcs.
c. Visualization: The precipitin arcs that form after immunodiffusion allow for the
identification and characterization of specific proteins based on their reaction with
the specific antibodies.

67. Working mechanism
The working mechanism of IEP is as follows:
1. A sample of proteins is loaded into wells in an agarose gel.
2. The gel is placed in an electrophoresis chamber and an electric field is
applied.
3. The proteins in the gel will migrate towards the oppositely charged electrode.
4. The proteins will be separated based on their electrical charge and size.
5. After electrophoresis, the gel is removed from the chamber and antibodies
that are specific for the proteins in the sample are added.
6. The antibodies will diffuse through the gel and react with the antigens.
7. The antibodies and antigens will form a precipitate at the point where they
meet.
8. The precipitate can be visualized by staining the gel.

68. Advantages
The advantages of IEP include:
• It is a sensitive technique that can be used to
detect even small amounts of proteins.
• It is a versatile technique that can be used to
identify a wide variety of proteins.
• It is a relatively simple technique that can be
performed in most laboratories.

69. Limitations
a.Semi-quantitative: Immunoelectrophoresis is not a fully
quantitative technique, and the intensity of bands may not
directly correlate with protein concentrations.
b. Labor-intensive: The technique can be time-consuming,
particularly when analyzing multiple samples or proteins.
c. Low resolution: Compared to modern techniques like
Western blotting or ELISA, immunoelectrophoresis has
relatively low resolution and sensitivity.

70. Applications
a. Clinical diagnostics: Immunoelectrophoresis is used to detect
and characterize serum proteins, including immunoglobulins,
complement proteins, and acute-phase reactants, for diagnostic
purposes.
b. Immunology research: It is employed in various immunological
studies to identify and analyze proteins involved in immune
responses.
c. Quality control in biopharmaceuticals: Immunoelectrophoresis is
used to monitor the composition and purity of biopharmaceutical
products, such as antibodies and vaccines.
d. Blood banking: The technique is utilized in blood typing and
antibody screening for safe blood transfusions.

71. Example
An example of the use of IEP is in the diagnosis of
multiple myeloma. Multiple myeloma is a cancer of
plasma cells, which are a type of white blood cell
that produces antibodies. In multiple myeloma, the
plasma cells produce an abnormal type of antibody
called a monoclonal protein. This protein can be
detected by IEP.

72. IEF

73. Basic principle
Isoelectric Focusing relies on the principle that charged
molecules, such as proteins, will migrate in an electric field
until they reach a pH at which they have no net charge, i.e.,
their pI. In an IEF setup, a pH gradient is created in a gel
matrix, typically using immobilized pH gradients (IPG strips).
Proteins are loaded onto the gel, and an electric field is
applied. Proteins then move within the gel matrix towards the
pH region that corresponds to their pI and become focused
in sharp bands at their respective isoelectric points.

75. Instrumentation
IEF can be performed using different equipment, but the
most common setups include:
Flatbed IEF: Where the gel is placed on a flat support and
the electric field is applied using electrodes at each end of
the gel.
b. Capillary IEF: Where the gel is placed in a capillary tube
and the electric field is applied across the length of the
capillary.

76. Working mechanism
a.Creating the pH gradient: This is achieved by using IPG strips, which contain a
series of immobilized buffering groups with different pKa values. When an
electric field is applied, these buffering groups create a stable pH gradient across
the gel.
b. Sample loading: The protein mixture is loaded onto the gel, usually at the
acidic end of the pH gradient.
c. Application of electric field: An electric field is applied across the gel, causing
the proteins to migrate towards their pI.
d. Isoelectric focusing: As the proteins reach their pI, they stop migrating and
become focused into narrow bands.

77. Advantages
a.High resolution: IEF can separate proteins with very
similar charges and resolve complex mixtures with high
precision.
b. Sensitivity: It can detect proteins present in minute
quantities.
c. Sample preservation: IEF is a gentle technique that
preserves protein structure and activity.
d. Automation: The process can be automated, making
it suitable for high-throughput analysis.

78. Limitations
a.Time-consuming: IEF can be a relatively slow process
compared to other techniques.
b. Limited pH range: The pH range of the gel is a crucial
factor, and it may not cover the pI values of all proteins of
interest.
c. Sample complexity: Overlapping protein bands may occur
in complex mixtures, making interpretation challenging.

79. Applications
a.Protein analysis: IEF is widely used for protein profiling,
post-translational modification analysis, and protein purity
assessment.
b. Proteomics: It plays a crucial role in 2D gel
electrophoresis, a common method for protein separation.
c. Medical diagnostics: IEF is used in clinical laboratories to
detect protein abnormalities in diseases like cancer and
genetic disorders.
d. Biotechnology: It is employed for protein purification in
research and biopharmaceutical production..

80. 2D Electrophoresis

81. Basic principle
The basic principle of 2D electrophoresis involves performing two
consecutive separations on the same sample. In the first dimension,
proteins are separated based on their isoelectric point using isoelectric
focusing (IEF). This creates a pH gradient in a gel matrix, and proteins
migrate to their respective pI positions. In the second dimension, the
proteins from the first dimension gel are then subjected to SDS-PAGE
(sodium dodecyl sulfate polyacrylamide gel electrophoresis) based on
their molecular weight. This results in a two-dimensional protein
separation pattern, which allows for the resolution of a large number of
proteins in a single gel.

82. Instrumentation
The instrumentation required for 2D electrophoresis includes
electrophoresis apparatus for both IEF and SDS-PAGE.
Specialized equipment is needed for IEF, such as isoelectric
focusing chambers and immobilized pH gradient (IPG) strips.
For SDS-PAGE, standard electrophoresis apparatus and
polyacrylamide gels are used.

84. Instrumentation
The working mechanism of 2D electrophoresis involves the following
steps:
● a. Isoelectric Focusing (IEF): In the first dimension, proteins are
loaded onto a gel strip with a pH gradient. An electric field is applied,
and proteins migrate along the pH gradient until they reach the region
of the gel with a pH equal to their isoelectric point. At this point, the
proteins become focused in narrow bands according to their pI.
● b. SDS-PAGE: The gel strip containing the focused proteins from the
first dimension is then placed on top of an SDS-PAGE gel. An electric
field is applied again, causing the proteins to separate based on their
molecular weight. SDS denatures the proteins and imparts a negative
charge, so the migration in this dimension is determined primarily by
size.

85. Advantages
a.High resolution: 2D electrophoresis allows for the
separation of a large number of proteins, enabling the
detection of minor protein variants and isoforms.
b. Improved sensitivity: The two-step separation process
enhances sensitivity, especially for low-abundance proteins.
c. Identification and characterization: The separated proteins
can be further analyzed using various staining methods or
transferred to a membrane for Western blotting or mass
spectrometry-based identification.

86. Limitations
a.Labor-intensive: 2D electrophoresis can be time-
consuming and requires careful handling of gels to prevent
artifacts.
b. Limited dynamic range: The detection of very high-
abundance and very low-abundance proteins in the same
gel may be challenging due to the limited dynamic range of
the technique.
c. Protein modifications: Post-translational modifications,
such as glycosylation, may affect protein migration and
complicate the interpretation of results.

87. Applications
a.Proteomics: 2D electrophoresis is a key tool in proteomics
research for protein profiling and identifying changes in
protein expression under different conditions.
b. Disease biomarker discovery: It is used to identify
potential biomarkers associated with diseases like cancer
and neurological disorders.
c. Comparative analysis: 2D electrophoresis is employed to
compare protein profiles between different samples or
experimental groups to study disease mechanisms or drug
effects.