Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide
3. • 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
4. 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:
Netcharge
Charge/massratio,
Molecularshape and
The temperature, porosity and viscosity of the matrix through
which the molecule migrates.
5. 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
postelectrophoresisstain can be applied.
6. • 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.
• Different types of gels which can be used are; Agar
and Agarose gel, Starch, Sephadex, Polyacrylamide
gels.
7. 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.
8. 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.
9. Instrument and reagents
1. Electrophoresis apparatus
2. Buffer
3. Power supply
4. Supporting media
5. Detection and Quantification
12. 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).
13. 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
14. 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
15. ⚫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)
16. Agarose
Polysaccharide
extracted from sea
weed.
Gel casted
horizontally
Non-toxic.
Separate large
molecules
Commonly used for
DNA separations.
Staining can be done
before or pouring the
gel.
Polyacrylamide
gel
Cross-linked polymer
of acrylamide.
Gel casted vertically.
Potent neuro-toxic.
Separate small
molecules.
Used for DNA or
protein separations.
Staining can be done
after pouring the gel.
Agarose
Polyacrylamide Gel
20. Gel electrophoresis
- electrode + electrode
DNA fragments
Agarose gel
~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
- electrode + electrode
current
~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
21. 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.
23. Agarose gel electrophoresis
Commonly used support medium
Less expensive than cellulose acetate
Equally good separation
Agar is a complex acidic polysaccharide containing
monomers of sulfated galactose
Agarose is a sulfate free fraction of Agar
Gel is prepared in buffer and spread over a microscopic slide
A small sample of serum or biological fluid is applied by
cutting in to the gel with a sharp edge
The electrophoretic rum takes about 90 minutes
24. ADVANTAGES
Easy to prepare and small concentration of agar is
required.
Resolution is superior to that of filter paper.
Large quantities of proteins can be separated and
recovered.
Adsorption of negatively charged protein molecule is
negligible.
It adsorbs proteins relatively less when compared to other
medium.
Sharp zones are obtained due to less adsorption.
Recovery of protein is good, good method for preparative
purpose.
25. Factors affecting separation in gel
electrophoresis
⚫Thesample:
⚫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
fibrousones.
26. ⚫The electric field: An increase in the potential
gradient increases the rateof migration.
⚫The medium: The inert medium can exert
adsorption or molecular sieving effects on the
particle influencng its rateof migration.
⚫Adsorption: retention of the component on the
surfaceof supporting medium.
⚫Molecular sieving: media such as
polyacrylamide, sephadex have cross
agar,
linked
structures giving rise to pores within the gel
beads.
27. ⚫The buffer: the buffercan affect theelectrophoretic
mobility by:
⚫Ionic strength: increase in ionic strength of buffer
means a larger share of current is carried by bufferand
smaller proportion by sample, while decrease in ionic
strength isvice-versa.
⚫pH: pH determines the degree of ionization of organic
compounds. Where ionization is inversely proportional
to pH.
28. 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
