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GEL ELECTROPHORESIS
Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins
according to molecular size. Charged molecules move through a gel when an electric current is passed
across it. The movement of charged molecules is called migration.
TYPES OF GEL
1. Agarose
Agarose gels are made from the natural polysaccharide polymers extracted from seaweed.
Agarose gels are easily cast and handled compared to other matrices, because the gel setting is
a physical rather than chemical change. Samples are also easily recovered. After the experiment
is finished, the resulting gel can be stored in a plastic bag in a refrigerator.
2. Polyacrylamide
Used to separate most proteins and small oligonucleotides because of the presence of small
pores. Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used
polyacrylamide gels to separate DNA fragments.
3. Starch
Partially hydrolyzed potato starch makes for another non-toxic medium for protein
electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured
proteins can be separated according to charge and size.
Preparing the gel
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Agarose gels are typically used to visualize fragments of DNA. The concentration of agarose used
to make the gel depends on the size of the DNA fragments you are working with.
The higher the agarose concentration, the denser the matrix and vice versa.
To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high
temperature.
The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make
wells for the sample to be pipetted into.
Once the gel has cooled and solidified the comb is removed.
The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the
tank until the surface of the gel is covered.
Running the sample (Agarose gel)
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Add loading buffer to each of your digest samples.
Once solidified, place the agarose gel into the gel box (electrophoresis unit).
Fill gel box with 1xTAE (or TBE) until the gel is covered.
Carefully load a molecular weight ladder into the first lane of the gel.
Carefully load your samples into the additional wells of the gel.
6. Run the gel at 80-150V until the dye line is approximately 75-80% of the way down the
gel.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully
remove the gel from the gel box.
8. Using any device that has UV light, visualize your DNA fragments.
SDS-PAGE
Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used biotechnology to separate
biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.
For proteins, sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein samples to linearize
proteins and to impart a negative charge to linearized proteins. This procedure is called SDS-PAGE.
PRINCIPLE: Any charged ion or molecule migrates when placed in an electric field.
PRINCIPAL COMPONENTS OF SDS-PAGE:
The components of an SDS PAGE gel electrophoresis system are the following:
1. A Slab holder for vertical or horizontal gels (thin, flat sheets of many individual lanes)
2. Polyacrylamide or agarose gels (cm x cm x mm); these are poured for each analysis
3. Gel is amended with SDS to dissociate & charge proteins.
4. High voltage power supply (0.1-6 kV)
5. A detection technique (dye staining, fluorescence, or autoradiography to image separated
bands)
TWO DIMENSIONAL GELS
Two-dimensional gel electrophoresis is a form of gel electrophoresis commonly used to analyze
proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels.
The first dimension separates proteins according to their native isoelectric point (pI) using a form of
electrophoresis called isoelectric focusing (IEF). The second dimension separates by mass using ordinary
SDS-PAGE.
ISO ELECTRIC FOCUSING
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Isoelectric focusing (IEF) is a technique for separating different molecules by differences in their
isoelectric point.
Is ideal for separation of amphoteric substances.
Separation is achieved by applying a potential difference across a gel that contain a pH gradient.
It gives good separation with a high resolution compared to any other method.
APPLICATION OF GEL ELECTROPHORESIS
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DNA Analysis
Protein Analysis
Antibiotics Analysis
Vaccine Analysis
Analysis of PCR products