1. BY PALADI RAMYA
ROLL NO:-2020MSSB007
M. Sc SPORTS BIOCHEMISTRY
UNDER GUIDANCE OF
V..DINESH KUMAR
2. Electrophoresis ( Electro refers to the energy of electricity and Phrases, from Greek
verb phoros, means to carry across) is a technique for separating or resolving
charged molecules (such as amino acids, peptides, nucleotides, proteins, and
nucleic acids) in a mixture under the influence of an applied electric field.
Charged molecules in an electric field move or migrate, at a speed determined by
their charge to mass ratio .
According to the laws of electrostatics, an ion with charge ‘Q’ in an electric field
of strength ‘E’ will experience an electric force, F electrical .
The migration of the molecules depends on the size, shape & viscosity of the
medium.
For a spherical molecule it is given by the Stokes ‘law.
Stokes’ law(ℱ)=6Πηr
3. Moving boundary electrophoresis:-
* Is the electrophoresis in a free
solution, without a supporting
media.
*To separate the different charged
molecules present in a mixture, the
sample ( dissolved in a buffer
solution that serves as an electrolyte
and maintains the desired Ph) is
placed in a glass tube connected to
electrodes. *When an electrical
potential is applied across the tube,
the charged molecules migrate
toward one or the other electrode.
* Because different charged
molecules migrate at different rates,
a number of interfaces or
boundaries are formed between the
leading edge of each charged
molecules and the remaining
mixture.
Zone electrophoresis:-
*A sample is constrained to move in
some kind of inert matrix such as
filter paper moistened with buffer (
paper electrophoresis ) or a gel ( gel
electrophoresis ) .
* A matrix is required because the
electric current passing through the
electrophoresis solution generates
heat, which causes diffusion and
convective mixing of the bands in the
absence of a supporting matrix.
* The kind of supporting matrix used
depends on the type of molecules to
be separated and on the desired basis
for separation.
4. In electrophoresis, there are two primary factors that control how quickly a
particle can move and in what direction. First, the charge on the sample
matters. Negatively charged species are attracted to the positive pole of an
electric field, while positively charged species are attracted to the negative
end. A neutral species may be ionized if the field is strong enough.
Otherwise, it doesn't tend to be affected.
The other factor is particle size. Small ions and molecules can move
through a gel or liquid much more quickly than larger ones.
While a charged particle is attracted to an opposite charge in an electric
field, there are other forces that affect how a molecule moves. Friction and
the electrostatic retardation force slow the progress of particles through the
fluid or gel. In the case of gel electrophoresis, the concentration of the gel
can be controlled to determine the pore size of the gel matrix, which
influences mobility. A liquid buffer is also present, which controls the pH
of the environment.
5. As molecules are pulled through a liquid or gel, the
medium heats up. This can denature the molecules as
well as affect the rate of movement. The voltage is
controlled to try to minimize the time required to
separate molecules, while maintaining a good
separation and keeping the chemical species intact.
Sometimes electrophoresis is performed in a
refrigerator to help compensate for the heat.
6. *The electrophoresis mobility of small particles can be measured by micro
electrophoresis, where the time taken for small particles to traverse a known
distance is measured, or alternatively by a moving-boundary method. The
micro electrophoresis method has many advantages over the moving-
boundary method, and it is the more frequently adopted method.
*If the mobility of charged particles is examined in a micro electrophoresis
cell then it will be noticed that there is a whole range of velocities, with
some particles even moving in the opposite direction. This effect is due to
electro-osmotic flow within the cell. The true electrophoresis mobility can
only be determined at the ‘stationary levels’ where the electro-osmotic flow
is balanced by the hydrodynamic flow. The position of the stationary levels
depends on the shape of the micro electrophoresis cell, i.e. whether it is
circular or rectangular.
7.
*In the constant electric field, the force on charged molecule balances
each other;
*Where “QE “ and “Vʄ “ are given as electrical force and frictional
force.
Electrophoresis morbidity is the ratio of the migration rate of a
charged molecule to the applied electric field;
*So according to above equation, electrophoresis mobility is directly
proportional to the charge and inversely proportional to the viscosity
of the medium, size and shape of the molecule.
QE=Vʄ
μ=V/E=Q/ʄ
9. * This technique is useful for the separation of small charged molecules
such as amino acids and small proteins. A strip of filter paper is
moistened with buffer and the ends of the strip are immersed into
buffer reservoirs containing the electrodes.
The samples are spotted in the center of the paper, high voltage is
applied, and the spots migrate according to their charges. After
electrophoresis, the separated components can be detected by a
variety of staining techniques, depending upon their chemical
identity.
10. A new method, disc electrophoresis, t has been designed that
takes advantage of the adjustability of the pore size of a
synthetic gel and that automatically produces starting zones of
the order of 10 microns thickness from initial volumes with
thicknesses of the order of centimeters.
* 3- Gel system:
-small pore separating gel ( running gel )
-a larger pore spacer gel ( stacking gel )
-and a thin layer of large pore monomer solution ( sample gel
)-containing about 3μL of serum.
* The different composition cause discontinuities in the
electrophoresis matrix.
* During electrophoresis, all proteins migrate easily through
the large-pore gels and stack up on the separation gel in a very
thin zone.
11. *This improves resolution and concentrates protein components
at the border ( or starting zone ).
*Separation occurs at the bottom separation gel by the
molecular sieve phenomenon.
12. * Slab gel electrophoresis is a primary method used in clinical
chemistry lab.
* It has ability to simultaneously separate several samples in one run.
*It uses a rectangular gel regardless of thickness.
*Gels are cast on sheets of plastic backing.
*It is useful in separation of serum proteins,
isoenzymes,liporoteins,hemoglobin&fragments of DNA &RNA.
13. *TECHNIQUES:-
*Electrophoresis encompasses several related analytical
techniques. Examples include
Affinity electrophoresis - Affinity electrophoresis is a type of
electrophoresis in which particles are separated based on complex
formation or biospecific interaction.
*What makes it special is its ability to detect affinity interactions be it
in free or immobilized form. It can detect peptides and proteins.
*It is also vital in detecting small molecules, development of drugs,
and for immune-affinity activities.
APPLICATIONS:-
*Useful technique for separation of bimolecules such as plasma
proteins, enzymes, nucleic acids, lectins, receptors, and extracellular
matrix proteins by specific interactions with their ligands in electric
fields and for the determination of dissociation constants for those
interactions.
14. *Capillary electrophoresis is a type of electrophoresis used to separate
ions depending mainly on the atomic radius, charge, and viscosity. As
the name suggests, this technique is commonly performed in a glass
tube. It yields quick results and a high resolution separation.
ADVANTAGES:-
Ease of use—no gels to pour
Reusability—the polymer matrix can be reused (product-dependent)
Fast separation times
Better resolution (single base pair)
Automated reads—optical sequence reading
Higher throughput—multiple capillaries can be used simultaneously
Complete automation—a complete workflow (including CE) is available on
automatic genetic analysis systems
