Analytical Microbiology

1. INTRODUCTION TO
ELECTROPHORESIS

2. Electrophoresis
The term literally means “migration with electricity.”
It involves the separation of components of a sample
by the differential rate of migration of ions by
attraction or repulsion in an applied dc electric field.
The technique was first developed by ArneTiselius in
1930s for the study of serum proteins.
Electrophoresis is a method whereby charged molecules
in solution, chiefly proteins and nucleic acids, are
separated through migration in response to an electrical
field.

3. Electrophoresis is defined as the migration
of the charged particle through a solution
under the influence of an external electrical
field.
Ions that are suspended between two
electrodes tends to travel towards the
electrodes that bears opposite charges.
As an analytical tool, electrophoresis is
simple, rapid and highly sensitive.

4. Complex mixtures can be separated.
It can be used analytically to study the properties of a
single charged species or mixtures of molecules.
The relative mobility of individual molecules depends
on several factors, the most important of which are
net charge,
charge/mass ratio,
molecular shape and the temperature,
porosity and viscosity of the matrix through which
the molecule migrates.

5. PRINCIPLES OF ELECTROPHORESIS
• 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. However, different molecules will move at quite different
and individual rates depending on the physical characteristics of the
molecule
• The velocity of movement, v, of a charged molecule in an electric field
depends on variables described by Equation
v = E · q/f ----------- (i)
• where f is the frictional coefficient, q is the net charge on the molecule
and E.q is the force that drives the molecules towards an electrode.
• The frictional coefficient describes frictional resistance to mobility and
depends on a number of factors such as the mass of the molecule, its
degree of compactness, buffer viscosity and the porosity of the matrix in
which the experiment is performed.

6. Because the electrical field strength, E, may vary widely
between different experimental formats, the electrophoretic
mobility, μ, of a sample is defined by (Electrophoretic
mobility (μ ) is defined as the rate of migration (cm/s) per unit
field strength .)
μ = v/E -------------(iv)
Combining this with equation with equation (i) shows that;
μ = E · q/f .E·
= q/f ---------- (v)
That is, the ratio of net charge to frictional coefficient is the
basis of migration of the biomolecules in electrophoresis.
Since f is strongly mass dependent, therefore, mobility of
biomolecules depends on their charge/mass ratio.

7. In general, molecules will move faster
i) as their net charge increases,
ii) if the electric field strength increases, and
iii) as f decreases.
Molecules of similar net charge separate due to differences
in frictional coefficient,
Molecules of similar mass/shape may differ widely from
each other in net charge.
Consequently, it is often possible to achieve very high
resolution separation by electrophoresis.

8. General Procedure
General operations performed in conventional electrophoresis
include
(1) separation,
(2) staining,
(3) detection, and
(4) quantification.
In addition, several electrophoretic "blotting“ techniques have been
developed.

9. Components of Electrophoresis System:

11. Components of Electrophoresis System:
Two buffer chamber, each chamber contains an electrode made
of either platinum or carbon, the polarity of which is determined
by the mode of connection to the power supply.
The most commonly-used buffer systems in electrophoresis of
biomolecules are Tris-Borate-EDTA, Tris-Cl or Tris-glycine. Buffers are
held in reservoirs connected to each electrode and provide a constant
supply of ions to the electrophoresis system throughout the separation.
The electrophoresis support on which separation takes place may
contact the buffer directly or indirectly.
The entire apparatus is covered to minimize evaporation and
protect the system
The system is powered by a direct current/power supply unit.

13. Electrophoresis
• Electrophoresis is usually done with gels formed
in tubes, slabs (vertical gel), or on a flat bed
(horizontal gel).
• In many electrophoresis units, the gel is mounted
between two buffer chambers containing separate
electrodes, so that the only electrical connection
between the two chambers is through the gel.

14. In most electrophoresis units, the gel is mounted between two
buffer chambers containing separate electrodes so that the
only electrical connection between the two chambers is
through the gel.

15. Reactions in Cathode and Anode

16. Slab Gel Unit (Usually polyacrylamide)

17. Slab Gel Unit (Usually polyacrylamide)

18. Flat Bed Unit/horizontal (Usually Agarose)

19. Tube Gel Units (Usually polyacrylamide)

20. Power Supply Unit
The function of a power supply in an electrophoretic process
is to supply electrical power. Commercially available power
supplies allow operation under conditions of constant
(1)current, (2) voltage, or (3) power,
all of which are adjustable.

21. Role of the Solid Support Matrix
• It inhibits convection and diffusion, which would
otherwise impede separation of molecules
• It allows a permanent record of results through
staining after run
• It can provide additional separation through molecular
sieving
Solid support matrix
In electrophoresis through which the molecule migrates and
on which separation takes place is solid support matrix.
It may contact the buffer directly or indirectly.
Solid matrix used in electrophoresis:
• Paper (Filter), Starch, Cellulose acetate, Polyacrylamide and
Agar/Agarose

22. Solid matrix
•Supporting medium
Supporting medium is a matrix in which the protein separation takes place.
•Various type has been used for the separation either on slab or capillary form.
• Separation is based on to the charge to mass ratio of protein depending on the
pore size of the medium, possibly the molecular size.
•Properties:
•Chemical nature inert
•Availability easy
•Electrical conductivity high
•Adsorptivity low
•Sieving effect desirable
•Porosity controlled
•Transparency high
•Electro-endosmosis (EEO) low
•Rigidity moderate to high
•Preservation feasible
•Toxicity low
• Preparation easy

23. Buffer
A buffer is a solution characterized by the ability to resist the
change in pH when limited amount of acid or base are added
to it.
•Buffers are held in reservoirs connected to each
electrode and provide a constant supply of ions to the
electrophoresis system throughout the separation.
•The buffer in electrophoresis has two fold purpose:
1.Carry applied electrical current
2.They set the pH as which electrophoresis is carried
out.
• Thus they determine;
• Type of charge on solute.
• Extent of ionization of solute
• Electrode towards which the solute will migrate.
•The buffer ionic strength will determine the thickness
of the ionic cloud.

24. Commonly buffers used;
• Buffer pH value
•Phosphate buffer around 7.0
•Tris-Borate-EDTA buffer (TBE) around 8.0
•Tris-Acetate EDTA buffer (TAE) above 8.0
•Tris Glycine buffer (TG) more than 8.5
•Tris -Citrate-EDTA buffer (TCE) around 7.0
•Tris -EDTA buffer (TE) around 8.0
•Tris -Maleic acid -EDTA buffer (TME) around 7.5
•Lithium Borate - buffer (LB) around 8.6

25. Commonly buffers used;
• Buffer pH value
•Phosphate buffer around 7.0
•Tris-Borate-EDTA buffer (TBE) around 8.0
•Tris-Acetate EDTA buffer (TAE) above 8.0
•Tris Glycine buffer (TG) more than 8.5
•Tris -Citrate-EDTA buffer (TCE) around 7.0
•Tris -EDTA buffer (TE) around 8.0
•Tris -Maleic acid -EDTA buffer (TME) around 7.5
•Lithium Borate - buffer (LB) around 8.6

26. •Gel electrophoresis is a laboratory method used
to separate mixtures of DNA, RNA, or proteins
according to molecular size.
•In gel electrophoresis, the molecules to be
separated are pushed by an electrical field through
a gel that contains small pores.
•Separation is brought about through molecular
sieving technique, based on the molecular size of
the substances.
Gel electrophoresis

27. •Gel material acts as a "molecular sieve”.
• Gel is a colloid in a solid form (99% is water).
• It is important that the support media is electrically neutral.
•A porous gel acts as a sieve by retarding or, in some cases,
by completely obstructing the movement of macromolecules
while allowing smaller molecules to migrate freely.
During electrophoresis, macromolecules are forced to move
through the pores when the electrical current is applied.
•Different types of gels which can be used are;
•Agar and Agarose gel, Starch,
•Sephadex, Polyacrylamide gels.

29. There are two types of gel electrophoresis:
Agarose and Polyacrylamide
• Although agarose and polyacrylamide differ greatly in
their physical and chemical structures, they both
make porous gels.
• A porous gel acts as a sieve by retarding or, in some
cases, by completely obstructing the movement of
macromolecules while allowing smaller molecules to
migrate freely.
• By preparing a gel with a restrictive pore size, the
operator can take advantage of molecular size
differences among proteins or nucleic acids.

30. Agarose and Polyacrylamide
• Because the pores of an agarose gel are large, agarose is
used to separate macromolecules such as nucleic acids,
large proteins and protein complexes
• Polyacrylamide, which makes a small pore gel, is used to
separate most proteins and small oligonucleotides.
• Both (agarose and polyacrylamide) are relatively
electrically neutral

31. Agarose Gels
• Agarose is a highly purified uncharged polysaccharide
derived from agar
• Agarose is chemically basic disaccharide repeating units of 3,6-
anhydro-L-galactose and galactose.
• Agarose dissolves in water when added to boiling liquid. It
remains in a liquid state until the temperature is lowered to
about 40°C at which point it starts to form gels
• Agarose gels are actually hydrocolloids, and they are held
together by the formation of weak hydrogen and
hydrophobic bonds
• The pore size may be predetermined by adjusting the
concentration of agarose in the gel, higher concentration of
agarose results smaller pore size.

32. Structure of the Repeating Unit of Agarose:
1,3-β-d-galactose and 1,4 α-L-3,6-anhydro-galactose
Basic
disaccharide
repeating units
of agarose,
G: 1,3-β-d-
galactose
and
A: 1,4-α-L-3,6-
anhydrogalactos
e

37. • ADVANTAGES:
1. Easy to prepare and small concentration of agar is
required.
2. Resolution is superior to that of filter paper.
3. Large quantities of proteins can be separated and
recovered.
4. Adsorption of negatively charged protein molecule is
negligible.
5. It adsorbs proteins relatively less when compared to other
medium.
6. Sharp zones are obtained due to less adsorption.
7. Recovery of protein is good, good method for preparative
purpose.

38. DISADVANTAGES:
• Electro osmosis is high.
• Resolution is less compared to polyacrylamide
gels.
• Different sources and batches of agar tend to
give different results and purification is often
necessary.
APPLICATION:
Widely used in Immuno electrophoresis.

39. • POLYACRYLAMIDE GEL ELECTROPHORESIS
(PAGE)
•It is prepared by polymerizing acryl amide monomers
in the presence of methylene-bis-acrylamide to cross
link the monomers.
• Structure of acrylamide (CH2=CH-CO-NH2) •
• Polyacrylamide gel structure held together by
covalent cross-links.

40. • Polyacrylamide gels are tougher than agarose gels.
• It is thermostable, transparent, strong and relatively
chemically inert.
• Gels are uncharged and are prepared in a variety of
pore sizes.
• Proteins are separated on the basis of charge to
mass ratio and molecular size; this phenomenon is
called Molecular sieving.
ADVANTAGES:
•Gels are stable over wide range of pH and
temperature.
•Gels of different pore size can be formed.
•Simple and separation speed is good comparatively.

41. Crosslinking Acrylamide Chains

42. •Polyacrylamide Gel Electrophoresis (PAGE)
•a) The gel is poured vertically between two glass
plates.
•b.) Protein bands are separated on the basis of
relative molecular weight and visualized with stains.
• PAGE Procedure :
• The Polyacrylamide gel of different pore sizes is
cast as thin rectangular slab inside a plastic frame or
into a column inside a vertical tube.
• This is made often with large pore gel at the top and
small pore gel at the bottom and this slab / column is
placed vertically on a buffer solution taken in a
reservoir.

43. •Microgram quantity of samples dissolved in dense sucrose
solution or glycerol are placed in separate wells and are
covered by the same buffer solution having such a pH so as to
change sample components into anions.
•The foot of the gel is made to dip in the same buffer in the
bottom reservoir.
Cathode and anode are kept above and below the gel to impose
an electric field through the column.
•Macromolecular anions move towards the anode down the gel
column.
•There is no external solvent space, all the migratory particles
have to pass through the gel pores.
•Rate of migration depends on the charge to mass ratio.
Different sample components get separated into discrete
migratory bands along the gel column on the basis of
electrophoretic mobility and gel filtration effect.

45. Range of separation of DNA and proteins in
different concetration of polyacrylamide gels

46. •TYPES OF PAGE
•PAGE can be classified according the separation
conditions into:
•Native- PAGE /Non-denaturing PAGE
•SDS-PAGE/ Denaturing PAGE
•NATIVE-PAGE:
•Native gels are run in non-denaturing conditions,
so that the analyte's natural structure is maintained.
• Separation is based upon charge, size, and shape
of macromolecules.
• Useful for separation or purification of mixture of
proteins.
•This was the original mode of electrophoresis.

47. NONDENATURING ELECTROPHORESIS
Polyacrylamide Nondenaturing Electrophoresis
The gel formed by polyacrylamide is suitable for the electrophoretic
separation of proteins in their native state, that is under
nondenaturing conditions.
In such conditions, the protein is regarded as being in its intact fully-
active form with the correct secondary, tertiary and quaternary
structure.
It separates on the basis of intrinsic charges on amino acid side-chains
and other groups located on the protein surface. Since the precise
number and strength of positive and negative charges will vary from
protein to protein, each will have a characteristic mobility in a
nondenaturing system determined by a combination of these charges
together with physical characteristics of the protein such as mass and
shape.

48. DENATURED-PAGE OR SDS-PAGE:
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in biochemistry,
forensics, genetics and molecular biology to separate proteins
according to their electrophoretic mobility.
Native protein is unfolded by heating in the presence of
mercaptoethanol and SDS. SDS binds to the protein so that it
stays in solution and denatures.
Large polypeptides bind more SDS than small polypeptides, so
proteins end up with negative charge in relation to their size.
Separation is based upon the molecular weight of proteins.
The common method for determining MW of proteins.
Very useful for checking purity of protein samples.

49. •SDS is an anionic detergent which denatures
secondary and non–disulfide–linked tertiary
structures by wrapping around the polypeptide
backbone.
•In so doing, SDS confers a net negative charge to
the polypeptide in proportion to its length.
• Molecules in solution with SDS have a net negative
charge within a wide pH range.
• A polypeptide chain binds amounts of SDS in
proportion to its relative molecular mass.
•The negative charges on SDS destroy most of the
complex structure of proteins, and are strongly
attracted toward an anode in an electric field.

50. When treated with SDS and a reducing agent, the
polypeptides become linear chain of negative
charges with equal “charge densities" or charge per
unit length.
Therefore, in SDS polyacrylamide gel, migration of
protein is determined not by intrinsic electrical
charge of polypeptides but by molecular weight.

51. SDS PAGE analysis of protein under reducing conditions

52. •VISUALIZATION
• After the electrophoresis is complete, the molecules
in the gel are stained to make them visible.
• Ethidium bromide, silver, or coomassie blue dye
may be used for this process.
•If the analyte molecules fluoresce under ultraviolet
light, a photograph can be taken of the gel under
ultraviolet lighting conditions.
• If the molecules to be separated contain
radioactivity added for visibility, an autoradiogram can
be recorded of the gel.

53. Commonly used stains for detection of proteins
Stains Detection limit
Ponceau Red 1-2 mg
Amido Black 1-2 mg
Coomassie Blue 1.5 mg
India Ink 100 ng
Silver stain 10 ng
Colloidal gold 3 ng
Stains used for the detection of DNA is Ethidium bromide
(limit 10ng), it intercalates between the nitrogenous bases, it is
highly fluorescent compound, so under UV light the DNA will be
visualized.

54. Coomassie Blue Staining

55. Silver Staining

56. Continuous and Discontinuous Buffer Systems
• A continuous system has only a single separating gel
and uses the same buffer in the tanks and the gel
• In a discontinuous system a nonrestrictive large pore
gel, called a stacking gel, is layered on top of a
separating gel
• The resolution obtainable in a discontinuous system
is much greater than that obtainable in a continuous
one. However, the continuous system is a little easier
to set up

57. Continuous and Discontinuous Buffer
Systems

58. Isoelectric Point
• A certain pH at which there is no net charge on a
protein or amino acid is known as isoelectric point
(pI).
• Above its isoelectric point, a protein has a net
negative charge and migrates toward the anode in an
electrical field.
• Below its isoelectric point, the protein is positively
charge and migrates toward the cathode.
• At isoelectric pH proteins or amino acids will not
migrate in an electric field.

59. Isoelectric Focusing
• Isoelectric focusing is a method in which proteins are
separated in a pH gradient according to their isoelectric
points.
• Focusing occurs in two stages; first, the pH gradient gel is
prepared using a mixture of chemicals with different
isoelectric point.
• In the second stage, the proteins begin their migrations
toward the anode if their net charge is negative, or
toward the cathode if their net charge is positive
• When a protein reaches its isoelectric point (pI) in the pH
gradient, it carries a net charge of zero and will stop
migrating

61. Isoelectric focusing
Proteins are separated in pH gradient.
Protein migrate into the point where its net charge is zero –
isoelectric pH.
Protein is positively charged in solutions at pH values below its pI.
Protein is negatively charged in solution at pH above its pI.

62. •METHOD
•pH gradient is established in gel by addition of ampholytes
which increases the pH from anode to cathode.
• A protein mixture is placed in a well on the gel.
• With an applied electric field, proteins enter the gel migrates
until each reaches its pH equivalent to its (PI).
• Each species of proteins is thereby focussed into a narrow
band about its PI.
•The Anode of the column is connected to a reservoir
containing an acidic solution like phosphoric acid and Cathode
is connected to a reservoir containing alkaline solution like
sodium hydroxide.
On opening the two reservoir valves the two solutions are
allowed to diffuse into the column from their respective ends ,
setting up a PH gradient between the acidic anode and the
alkaline cathode.

63. •The valves are then closed and the current is switched on ,
causing the carrier ampholytes to migrate until they reach
the PH regions where they have no net charge.
•They will then remain stationary at these points.
•ADVANTAGES:
•1) As spreading of bands is minimized due to
application of the applied field and the PH gradient ,
high resolution can be achieved.
•2) Proteins that differ by as little as 0.001 PH units can
be separated.

64. Electrophoresis can be one dimensional or two dimensional.
When a mixtures of proteins are separated by two properties
in two dimensions then it is called two-dimensional
electrophoresis.
One dimensional electrophoresis is used for most routine
protein and nucleic acid separations.
Two dimensional separation of proteins is used for finger
printing, and when properly constructed can be extremely
accurate in resolving all of the proteins present within a cell.
Most common stabilizing media are polyacrylamide or agarose
gels.

65. Two-Dimensional Gel Electrophoresis
• Two-dimensional gel electrophoresis is widely used to separate
complex mixtures of proteins into many more components that
is not possible in conventional one-dimensional electrophoresis
• Each dimension separates proteins according to different
properties
• In the first dimension proteins are separated on the basis of
electro-focusing by tube gels
• In the second dimension proteins are separated on the basis of
molecular weight by SDS slab gel
• The analysis of 2-D gels is more complex than that of one-
dimensional gels because the components that show up as spots
rather than as bands.

67. •CAPILLARY ELECTROPHORESIS
•Capillary electrophoresis is an analytical technique that
separates ions based on their electrophoretic mobility .
• Capillary electrophoresis is a collection of wide range of
separation techniques which involve the application of high
voltages across buffer filled capillaries to achieve separations .
•The electrophoretic mobility is dependent upon the charge of
the molecule, the viscosity, and the atom's radius.
•The rate at which the particle moves is directly proportional to
the applied electric field--the greater the field strength, the
faster the mobility.
•Neutral species are not affected, only ions move with the
electric field. If two ions are the same size, the one with
greater charge will move the fastest.

68. For ions of the same charge, the smaller particle has less
friction and overall faster migration rate.
Capillary electrophoresis, is the technique of performing
electrophoresis in bufferfilled, narrow-bore capillaries, normally
from 25 to 100 mm in internal diameter (ID). Capillaries are
typically of 50 µm inner diameter and 0.5 to 1 m in length.
Many materials have been suggested and tested for the
construction of capillaries for CE. These include fused
silica, borosilicate glass, and polytetrafluoroethylene
(Teflon). Fused silica is now the preferred material for the
construction of capillaries.
The capillary is filled with electrolyte solution which conducts
current through the inside of the capillary. The ends of the
capillary are dipped into reservoirs filled with the electrolyte.
•Electrodes (platinum) are inserted into the electrolyte
reservoirs to complete the electrical circuit.

69. A high voltage (typically 10-30 kV) is applied.
• Due to electroosmotic flow, all sample components migrate
towards the negative electrode. The capillary can also be
filled with a gel, which eliminates the electroosmotic flow.
Separation is accomplished as in conventional gel
electrophoresis but the capillary allows higher resolution,
greater sensitivity, and on-line detection.
•Sample application is done by either
a)High voltage injection-potential is applied causing the
sample to enter capillary by combination of ionic attraction
and electroosmotic flow.
b)Pressure injection-pressure difference is used to drive the
sample into capillary by applying vaccum.

70. When PD is applied net migration occurs in the direction of
cathode.
• Even substance with net negative charge migrate in the
direction of cathode due to the phenomenon called as Electro
Osmotic Flow.
• Neutral molecule moves at the same speed as the EOF.
•Positively charged species move faster, speed is sum of
EOF and Electrophoretic mobility.
•Negatively charged molecules lag behind.
Capillary electrophoresis is used most predominately
because it gives faster results and provides high resolution
separation.
Capillary Electrophoresis (CE)

72. Application of Capillary Electrophoresis
Capillary electrophoresis is a modern separation technique, with vast
acceptance in the academic and industrial communities
DNA Fingerprinting
DNA fingerprinting is a useful tool for identifying the genotype of
living organisms by determining their DNA sequence. For this
technique, genomic DNA must be amplified by PCR. Capillary
electrophoresis separates this amplified DNA with a one base pair
resolution and creates specific peaks for each nucleotide to map the
DNA sequence.

73. PULSED FIELD GEL ELECTROPHORESIS
DNA in vivo is organized in extremely large single molecules which are arranged
in chromosomes. It is therefore necessary to process DNA by restriction digestion
to obtain sufficiently small molecules for separation and study in agarose or
polyacrylamide gels.
Sometimes it needs to study intact chromosomes or large DNA fragments of
chromosomes which exceed the resolution-range of these systems. An
electrophoretic technique called pulsed field gel electrophoresis has been
specifically developed for separations in the mass range 200–3000 kb.
In pulsed field gel electrophoresis, the direction of the field is varied continuously
during electrophoresis. This is achieved by alternately turning on and off the
current or pulsing the electrical field in short time intervals called the pulse time,
tp. Intact chromosomal DNA or large fragments of chromosomal DNA are
obtained by first lysing cells by treatment with a detergent or with enzymes such
as lysozyme. This treatment often generates fragments of chromosomal DNA due
to mechanical shearing.

74. Large DNA molecules are then loaded on agarose gels and electrophoresis is
initiated. An array of electrodes is arranged around the gel such that the
direction of electrophoresis may be continuously varied. Pulses of electricity are
passed through these electrodes. The direction of electrophoresis of DNA is
constantly alternated between the two electric field directions as the molecules
re-orientate themselves through a reorientation angle.
It is thought that, during reorientation, the helical structure is stretched and
then compressed. The time required for this to occur is the visceoelastic
relaxation time, tr, and it is dependent on molecular weight.
The ratio of tr to tp is crucial for good separation in pulsed field electrophoresis.
If tp is much shorter than tr then there is insufficient time for the molecule to
reorientate itself in response to the pulse. If tp is much larger than tr, then the
molecules will behave as in conventional agarose gel electrophoresis. When the
tr/tp ratio is between 0.1 and 1, optimal separation on the basis of mass is
achieved.

75. Figure : Pulsed field gel electrophoresis. DNA fragments are exposed to an
electric field which alternates in different directions as a result of electrical
pulses. The DNA moves through the gel in response to this field. Small molecules
move more quickly than large ones due to their shorter visceoelastic relaxation
times. For simplicity, an array of only four electrodes carrying equal charge is
shown. In reality, the array may consist of many electrodes arranged in various
geometries and carrying different proportions of the current thus generating a
complex electric field.

76. Applications of Pulsed Field Gel Electrophoresis
Because this technique facilitates analysis of DNA fragments on the scale of
individual chromosomes, it has found extensive uses in large-scale
mapping of chromosomes.
This has particular importance in bacterial taxonomy allowing the
identification of relationships between existing and novel strains of
bacteria.
In eukaryotes, pulsed field gel electrophoresis of yeast chromosomes has
revealed widespread length polymorphisms.
The technique has also been applied to studies on strand breaks in human
chromosomes as a result of exposure to toxic chemicals.