Electrophoresis is a technique used to separate charged particles such as proteins, nucleic acids, and other molecules based on their size and charge. It works by applying an electric field to encourage the particles to migrate through a gel or other medium at different rates. The document discusses the components, principles, and applications of electrophoresis, including different types of gels, buffers used, and methods of visualization. It is commonly used in fields like molecular biology, genetics, and forensics.
2. Definition
• The electrophoresis (from Latina phoresis-
move) is the instrument which measures
the migration of a charged particles under
the electric current toward anode
electrodes forming different sized bands.
• It is a separation technique, easy, simple
and highly sensitive test and used on body
fluids such as blood serum ,urine and CSF.
3. The electrophoresis purposes are :
1. To determine the number, amount and
mobility of components in a sample or to
separate them.
2. To obtain information about the electrical
double layers surrounding the particles.
3. Determination of molecular weight of
proteins and DNA sequencing.
5. Components of Electrophoresis unit
1. Consists of a high-voltage power supplier.
2. Positive and negative electrodes.
3. Buffer solution chamber.
4. And a support for the buffer such as filter paper,
cellulose acetate strips, polyacrylamide gel, or a
capillary tube.
5. Other components include dyes, combs, gel
mixtures, gel dryers and chemicals such as
denaturing agents, gel hardeners, and
ampholytes.
7. Principle of Electrophoresis
• In the electric field the negative end pushes
the ions or molecules through the gel, and a
positive end pulls the ions or molecules
through the gel.
• The larger molecules move more slowly than
smaller molecules.
• The different sized molecules form distinct
bands on the gel.
8. Principle of Electrophoresis
The rate velocity (V) of migration is according to
this equation;
F = frictional coefficient depends upon the size of the molecule not the shape.
E = electric field (Volt/ cm)
q = the net charge on molecule
Electrophoretic mobility µ= V/E or q/f
The µ is directly proportional to charge density
(charge/mass ratio)
V= Eq/F where
9. FACTORS AFFECTING
ELECTROPHORETIC MOBILITY
(a) The strength of electric field, size and shape.
(b) Relative hydrophobicity of the sample.
(c) Ionic strength and temperature of the buffer.
(d) Molecular size of the taken biomolecule.
(e) Net charge density of the taken bio molecule.
(f) Shape of the taken biomolecule.
10. FACTORS AFFECTING ELECTROPHORETIC
MOBILITY
1. Charge – higher the charge greater the
electrophoretic mobility.
2. Size – bigger the molecule, greater are the
friction exerted on it by the medium.
3. Shape – rounded contours elicit lesser
frictional and electrostatic retardation compared
to sharp contours. Therefore globular protein
move faster than fibrous protein
13. History
In 1807, a Russian Physicist, Alexander Reuss
observed a novel phenomenon - when electricity
was passed through a glass tube containing water
and clay, colloidal particles moved towards the
positive electrode.
In 1955, Oliver Smithies found that separation of
human tissue extracts with high resolution by starch
gel electrophoresis.
In between 1950 to 1970, an enumeration of
techniques and instrumentation for Electrophoresis
were developed.
14. Classification of Electrophoresis
1. Zone Electrophoresis (on supporting media)
A. Paper Electrophoresis, B. Cellulose
Electrophoresis. C. Thin layer Electrophoresis
and D. Gel Electrophoresis (SDS, PAGE, etc..)
2. Moving boundary electrophoresis such as;
A- Capillary Electrophoresis, B- Isotachophoresis
C- Isoelectric Focusing, D- Immuno
Electrophoresis
15. Paper Electrophoresis:
• A filter paper is immersed into buffer .
• The samples are spotted in the center of the
paper. then the separated components can be
detected by a variety of staining techniques.
• Applications: it is used for serum analysis for
low MW muscle protein (Myosin), egg protein
(albumin), milk protein (casein), snake and
insect venoms . It take 14-16 hours.
16.
17.
18. Cellulose Acetate Electrophoresis
A modified paper developed by Kohn in 1958.
Hydroxyl groups of cellulose (paper) converted
to acetyl groups- thus molecules become non-
adsorbing.
Non-toxic low sugar content so suitable for
electrophoresis of polysaccharides.
Applications: for separation of glycoproteins,
lipoproteins and hemoglobin from blood.
19. Starch gel electrophoresis
* Introduced by Smithies
(1955).
* Starch Cooking hydrolyzed
potato used as Supporting
media.
* 2 forms - α amylose
(unbranched) & amylopectin
(branched) polymers.
* Mostly used for protein
separation.
20. Gel Electrophoresis:
It uses the gel as supporting media, It is usually
performed for analytical purposes and to
partially purify RNA, DNA molecules .
(a) Vertical Gel Apparatus: It is used for the
separation of proteins in SDS-PAGE.
(b) Horizontal Gel Apparatus: It is used for
immune-electrophoresis, isoelectric focusing
and electrophoresis of DNA and RNA in the
agarose gel.
23. Gel used in Electrophoresis
• The term "gel" is a crosslinked polymer .
• When separating proteins or small nucleic
acids (DNA, RNA, or oligonucleotides) the gel is
usually composed of different concentrations
of acrylamide and a cross-linker.
• When separating larger nucleic acids (greater
than a few hundred bases), the preferred
matrix is purified agarose.
24. Limits of electrophoresis
1. Passing current through a gel causes heating,
gels may melt during electrophoresis.
2. Running for too long can exhaust the
buffering capacity of the solution.
3. Further, different preparations of genetic
material may not migrate consistently with
each other, for morphological or other
reasons.
25. Types of gel
• Polyacrylamide gels are
usually used for proteins,
and have very high
resolving power for small
fragments of DNA (5-500
bp).
• Polyacrylamide gels are run
in a vertical configuration.
• Agarose sets thermally.
• Agarose gels have lower
resolving power for DNA
fragments of usually 50-
20,000 bp in size.
• Agarose gels are typically
run horizontally in a
submarine mode
• Polyacrylamide forms in
a chemical
polymerization reaction
26. Buffers
• Buffers in gel electrophoresis are used to provide
ions to maintain the pH at a relatively constant
value.
• Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE).
• Borate is problematic.
• TAE has the lowest buffering capacity but
provides the best resolution for larger DNA. This
means a lower voltage and more time, but a
better product.
27. Visualization
• DNA may be visualized using ethidium
bromide which, when intercalated into DNA,
fluoresce under ultraviolet light, while protein
may be visualised using silver stain or
Coomassie Brilliant Blue dye.
• If the molecules to be separated contain
radioactivity, for example in a DNA sequencing
gel, an autoradiogram can be recorded of the
gel. Photographs can be taken of gels, often
using a Gel Doc system.
29. Applications of Gel Electrophoresis.
• Estimation of the size of DNA molecules
following restriction enzyme digestion, e.g. in
restriction mapping of cloned DNA.
• Analysis of PCR products, e.g. in molecular
genetic diagnosis or genetic fingerprinting.
• Gel electrophoresis is used in forensics,
molecular biology, genetics, microbiology and
biochemistry.
