Gel Electrophoresis

A note on the use of gel electrophoresis in biology.

1. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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Gel Electrophoresis
Gel Electrophoresis is a method for separation and analysis of macromolecules (DNA,
RNA, and proteins) and their fragments, based on their size and charge. It is used to
separate a mixed population of DNA and RNA fragments by length, to estimate the
size of DNA and RNA fragments, or to separate proteins by charge or size.
PRINCIPLE OF ELECTROPHORESIS
Charged molecules move to their counter charge electrodes but the electric field is
removed before they reach the electrode. Movement of charged species in an electric
field gives differential mobility to the sample based on the charge and hence resolves
them. The movement of charged particles is retarded with the addition of a polymeric
gel so that sufficient time is available for resolving the sample. The polymeric gel is
inert, uncharged, and does not bind with the sample molecules. Instead, it forms pores
of different sizes (depending on the concentration of the polymer) and the sample
passes through these pores, as a result, their electrophoretic mobility is reduced.
TYPES OF GEL ELECTRO PHORESIS
Following are the types of gel electrophoresis:
 Agarose Gel Electrophoresis: It is a method of gel electrophoresis used to separate
a mixed population of macromolecules such as DNA in a matrix of agarose gel
(which is made from the natural polysaccharide polymers extracted from seaweed).
 Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis (SDS – PAGE): It
is used for separating proteins due to the uniform pore size provided by the
polyacrylamide gel.
 Starch Electrophoresis: It is a method for separating non-denatured proteins in a
partially hydrolyzed potato starch medium. The results are visualized using Napthal
Black or Amido Black staining. Typical starch gel concentrations are 5% to 10%.
AGAROSE GEL ELECTROP HORESIS (DNA SEPERATION)

2. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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Agarose gel electrophoresis is widely used to separate DNA (or RNA) of varying sizes
that may be generated by restriction enzyme digestion or by other means, such as
PCR.
Materials: The materials required for agarose gel electrophoresis are as follows:
 Electrophoresis Chamber: This is the tank in which the process takes place, it is
connected to positive and negative electrodes (which in turn are connected to a
power supply).
 Comb: A comb is placed in the liquid agarose after it has been poured. Removing
the comb from the hardened gel produces a series of wells used to load the DNA.
 Gel Casting Tray: These trays are available in a variety of sizes and are composed
of UV-transparent plastic. The open ends of the trays are closed with tape while
the gel is being cast, then removed before electrophoresis.
 Agarose Gel: Agarose is a polysaccharide, generally extracted from certain red
seaweed. Agarose gel has a large pore size and good gel strength, making it
suitable as an anti-convection medium for the electrophoresis of DNA and large
protein molecules.
 Buffer: During electrophoresis, water undergoes hydrolysis (H2O → H
+
+ OH
-
).
Buffers prevent the pH from changing by reacting with the H
+
or OH
-
products. The
most common buffer used is called TRIS – [tris(hydroxymethyl)aminomethane].
 Staining Agent (Dye): To make DNA fragments visible after electrophoresis, the
DNA must be stained. Ethidium bromide is the best choice, when it binds to DNA
it fluoresces under ultraviolet light, hence allowing visualization.
 DNA Ladder: It is a solution of DNA molecules of different lengths. DNA ladder
consists of known DNA sizes used to determine the size of an unknown DNA
sample (hence serving as a reference).
 Sample DNA: This is the sample that has to be separated.
Procedure: Due to its negatively charged backbone, DNA is strongly attracted to a
positive electrode. In agarose gel electrophoresis, the gel is oriented horizontally in a

3. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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buffer solution. Samples are loaded into sample wells on the side of the gel closest to
the negative electrode, then drawn through the molecular sieve of the agarose matrix
toward the positive electrode. The agarose matrix impedes the movement of larger
molecules through the gel, whereas smaller molecules pass through more readily.
Thus, the distance of migration is inversely correlated to the size of the DNA fragment,
with smaller fragments traveling a longer distance through the gel. Sizes of DNA
fragments within a sample can be estimated by comparison to fragments of known
size in a DNA ladder also run on the same gel.

Figures: (a) The process of agarose gel electrophoresis. (b) A researcher loading
samples into a gel. (c) This photograph shows a completed electrophoresis run on an
agarose gel. The DNA ladder is located in lanes 1 and 9. Seven samples are located
in lanes 2 through 8. The gel was stained with ethidium bromide and photographed
under ultraviolet light.

4. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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Visualization: The locations of the DNA or RNA fragments in the gel can be detected
by various methods. One common method is adding ethidium bromide, a stain that
inserts into the nucleic acids at non-specific locations and can be visualized when
exposed to ultraviolet light. Other stains that are safer than ethidium bromide, a
potential carcinogen, are now available.
Applications: There are several situations in which a researcher might want to
physically separate a collection of DNA fragments of different sizes. A researcher may
also digest a DNA sample with a restriction enzyme to form fragments. The resulting
size and fragment distribution pattern can often yield useful information about the
sequence of DNA bases that can be used, much like a bar-code scan, to identify the
individual or species to which the DNA belongs.
SDS – PAGE (PROTEIN SEPARATION )
Sodium Dodecyl Sulphate – Polyacrylamide gel electrophoresis (SDS – PAGE) is an
electrophoretic technique used for separating proteins. In SDS – PAGE, the gel matrix
is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is
typically performed using a vertical gel apparatus.
Materials: The materials used in SDS – PAGE are as follows:
 Electrophoresis Chamber: This is the tank in which the process takes place, it is
connected to positive and negative electrodes (which in turn are connected to a
power supply).
 Comb: A comb is placed in the liquid polyacrylamide gel after it has been poured.
Removing the comb from the hardened gel produces a series of wells used to load
the protein.
 Gel Casting Tray: These trays are available in a variety of sizes and are composed
of UV-transparent plastic. The open ends of the trays are closed with tape while
the gel is being cast, then removed before electrophoresis.

5. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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 Polyacrylamide Gel: Polyacrylamide is a polymer formed from acrylamide subunits.
It is highly water-absorbent and forms a soft gel when hydrated.
 Sodium Dodecyl Sulphate: It is a negatively charged detergent which is used to
denature and coat proteins, it masks the native charges and allowing separation
based on size only.
 Protein Sample: This protein sample has to be separated, it is usually treated with
SDS.
 Protein Ladder: The reference protein ladder will allow the determination of the
location of protein of interest-based on its molecular size.
 Running Buffer: It allows the protein samples to run through the polyacrylamide
gel.
 Staining Buffer: Once the electrophoresis is over, SDS-PAGE gel is stained in
Coomassie Stain Solution.
 Destaining Buffer: As Coomassie stain also binds to SDS-PAGE gel, an SDS-
PAGE destain solution is used to destain the gel, so that only protein bands can
be visualized.
Procedure: For separation, the denatured samples are loaded onto a gel of
polyacrylamide, which is placed in an electrophoresis buffer with suitable electrolytes.
Thereafter, a voltage (usually around 100 V, 10-20 V per cm gel length) is applied,
which causes a migration of negatively charged molecules through the gel in the
direction of the positively charged anode. The gel acts like a sieve. Small proteins
migrate relatively easily through the mesh of the gel, while larger proteins are more
likely to be retained and thereby migrate more slowly through the gel, thereby allowing
proteins to be separated by molecular size. The electrophoresis lasts between half an
hour to several hours depending on the voltage and length of gel used.
Visualization: Following electrophoresis, proteins are visualized through staining,
commonly with either Coomassie blue or a silver stain.

6. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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Figures: (a) SDS is a detergent that denatures proteins and masks their native
charges, making them uniformly negatively charged. (b) The process of SDS-PAGE
is illustrated in these steps. (c) A photograph of an SDS-PAGE gel shows Coomassie-
stained bands where proteins of different sizes have migrated along the gel in
response to the applied voltage. A size standard lane is visible on the right side of the
gel.
Applications: The SDS-PAGE in combination with a protein stain is widely used in
biochemistry for the quick and exact separation and subsequent analysis of proteins.
It is mostly used for analytical purposes and less for preparative purposes, especially
when larger amounts of a protein are to be isolated. Additionally, SDS-PAGE is used

7. SYED MUHAMMAD KHAN ( BS HONS. ZOOLOGY)

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in combination with the western blot for the determination of the presence of a specific
protein in a mixture of proteins - or the analysis of post-translational modifications.
Post-translational modifications of proteins can lead to a different relative mobility (i.e.
a band shift) or to a change in the binding of a detection antibody used in the western
blot (i.e. a band disappears or appears). In mass spectrometry of proteins, SDS-PAGE
is a widely used method for sample preparation before spectrometry, mostly using in-
gel digestion. In regards to determining the molecular mass of a protein, the SDS-
PAGE is a bit more exact than an analytical ultracentrifugation but less exact than a
mass spectrometry or - ignoring post-translational modifications - a calculation of the
protein molecular mass from the DNA sequence. In medical diagnostics, SDS-PAGE
is used as part of the HIV test and to evaluate proteinuria (presence of excess proteins
in the urine).