Basic Principle, working mechanism, instrumentation,advantages,limitations and applications of Electrophoresis Moving boundary Paper AGE & PAGE

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. 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.

46. 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.

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

48. Polyacrylamide Gel electrophoresis

49. 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.

50. 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.

51. 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.

52. 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.

53. 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.

54. ● 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.

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