2. Positive or negative electrical charges are
frequently associated with biomolecules. When
placed in an electric field, charged biomolecules
move towards the electrode of opposite charge due
to the phenomenon of electrostatic attraction
Introduction:
3. Electrophoresis
Electrophoresis is the separation of charged
molecules in an applied electric field.
The relative mobility of individual molecule
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.
4. Gel Electrophoresis
Gel electrophoresis is a method for separation and
analysis of macromolecules like DNA, RNA and
proteins or their fragments, based on their size and
charge.
Gel electrophoresis uses a gel as an anti-convective
medium and/or sieving medium during
electrophoresis.
Gels suppress the thermal convection caused by
application of the electric field, gels can also simply
serve to maintain the finished separation, so that a
post electrophoresis stain can be applied.
5. Principle
By placing the substance to be separated in wells of the gel
and applying an electric current, allows the molecule to move
through the matrix at different rates towards the anode if
negatively charged or toward the cathode if positively
charged.
As they move through the gel, the larger molecules will be
held up as they try to pass through the pores of the gel, while
the smaller molecules will be impeded less and move faster.
This results in a separation by size, with the larger molecules
nearer the well and the smaller molecules farther away.
6. Principle of separation
According to charge: When charged molecules are
placed in an electric field, they migrate toward either
the positive (anode) or negative (cathode) pole
according to their charge.
According to size: The smaller molecules move more
swiftly than the larger sized ones, as the can travel
through the pores more easily than the later.
7. Instrument and reagents
1. Electrophoresis apparatus
2. Buffer
3. Power supply
4. Supporting media
5. Detection and Quantification
10. 2. Buffer:
Buffers in gel electrophoresis are used to provide ions
that carry a current and to maintain the pH at a
relatively constant value.
The most common being, for nucleic acids
Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA(TBE).
11. 3. Power supply:
• The electrodes are
connected to their
respective terminals of
the electrophoresis
chamber and to the
power supplier with
controls for rate of
current flow.
•The best resolution of
fragments larger than
about 2 kb is attained by
applying no more than 5
volts per cm to the gel
12. 4. Supporting media: (Gel)
1. Starch
2. Agar/agarose
3. Cellulose acetate
4. Polyacrylamide gel
The kind of supporting matrix used depends on type
of molecules to be separated and the desired basis for
separation: charge, molecular weight or both
13. Agarose and polyacrylamide gels are cross-linked,
spongelike structure
It is important that the support media is electrically
neutral. Presence of charge group may cause:
-Migration retardation
-The flow of water toward one or the other electrode
so called ‘Electroendosmosis (EEO)’, which decrease
resolution of the separation
Agarose Gels have fairly large pore sizes and are used
for separating larger DNA molecules (Restriction
Fragment Length Polymorphism Analysis)
Polyacrylamide Gels are used to obtain high resolution
separations for smaller DNA molecules (STR analysis
and DNA sequence analysis)
17. Gel electrophoresis
Agarose gel
- electrode + electrodeDNA fragments
buffer~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
- electrode + electrode
current
buffer~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
18. visualization
The molecules in the gel are stained to make them visible.
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.
SYBR Green I is more expensive, but 25 times more sensitive
and possible safer than ethidium bromide.
SYBR Safe is a variant of SYBR Green, and show low level of
mutagenicity and toxicity.
Other less frequently used markers are Cresol red and Orange
G.
19. Factors affecting separation in gel
electrophoresis
The sample:
The charge/mass ratio of the sample dictates its
electrophoretic mobility.
Charge: Higher the charge, greater the electrophoretic
mobility.
Size: Size is inversely proportional to electrophoretic
mobility.
Shape: Globular substances move faster than the
fibrous ones.
20. The electric field: An increase in the potential
gradient increases the rate of migration.
The medium: The inert medium can exert
adsorption or molecular sieving effects on the
particle influencng its rate of migration.
Adsorption: retention of the component on the
surface of supporting medium.
Molecular sieving: media such as agar,
polyacrylamide, sephadex have cross linked
structures giving rise to pores within the gel
beads.
21. The buffer: the buffer can affect the electrophoretic
mobility by:
Ionic strength: increase in ionic strength of buffer
means a larger share of current is carried by buffer and
smaller proportion by sample, while decrease in ionic
strength is vice-versa.
pH: pH determines the degree of ionization of organic
compounds. Where ionization is inversely proportional
to pH.
22. Applications
Separation of Deoxyribonucleic acid
Separation of ribonucleic acid
Separation of protein molecules
It may be used as preparative technique prior to use of other
methods such as mass spectroscopy, cloning, DNA Sequences,
Southern Blotting for further characterization.
Separation of amino acid
Separation of lipoproteins
Separation of enzyme in blood
Separation of antibiotic drug
23. References:
Instrumental methods of chemical analysis.
By Dr. B.K.Sharma, Page no. 661-670.
Instrumental analysis by William Kemp.
http://www.intechopen.com/books/gel-
electrophoresisprinciples-and-basics
Wikipedia .
