GEL ELECTROPHORESIS
Guided by : Sharma maa’m
Presented by :- Sima rathod
M. pharm first year (phamacognosy)
Government College of pharmacy,
Amaravati.
Introductio
n
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
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
The temperature, porosity and viscosity of the
matrix through which the molecule migrates.
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.
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.
Principle of separation
According to charge:
When charged molecules areplaced 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 moreswiftly than the larger
sized ones, as the can travel through the pores more
easily than the later.
Instrument and reagents
1.Electrophoresis apparatus
2.Buffer
3.Power supply
4.Supporting media
5.Detection and Quantification
1. Electrophoresis
apparatus
The casting tray is made up of glass or
plastic.
The comb contains varying number of teeth
in order to help in formation of well.
Electrophoresis apparatus
set up:
Electrophoresis chamber with buffer
solution Casting tray
Electrodes
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).
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
4. Supporting media: (Gel)
1. Starch
2. Agar/agarose
3. Cellulose acetate
4. Polyacrylamide ge
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
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)
5. Detection and
quantification:
Stains
• Protein staining
• Ethidium bromide staining
Blotting
• Southern blotting (for DNA)
• Northern blotting (for RNA)
• Western blotting (for protein)
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
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
thefibrous ones.
The electric field: An increase in the potential
gradient increases the rate of migration.
The medium: The inert medium can
exertadsorption 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.
The buffer:
the buffer can affect the electrophoretic mobility
by:
Ionic strength: increase in ionic strength of
buffermeans 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.
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
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
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