Gel Electrophoresis System- Apparatus, Parts, Types, Examples

Electrophoresis is a chemical process in which an electric charge in a solution flow

toward an opposing electrode. In the 1930s, Swedish biophysicist Arne Tisselius developed
electrophoresis while researching blood proteins. In 1948, Arne Tisselius received Novel
Prize in Chemistry for his contributions to the electrophoretic technique.

Gel electrophoresis is one of the laboratory methods for separating DNA, RNA, or
protein molecules based on their electric charge or size.
Principle of Gel Electrophoresis

The principle behind electrophoresis is the observation that the majority of

biomolecules exist as electrically charged particles with ionizable functional groups. A
solution containing biomolecules will have either positively or negatively charged ions
depending on the pH.

When charged molecules are placed in an electric field, they travel in the opposite
direction of the positive or negative pole. Depending on the mass and net charge of each
particle in the solution, ionized biomolecules will migrate at different rates when
exposed to an electric field. Negatively charged particles such as nucleic acids gravitate
toward the anode, while the positively charged particles toward the cathode. Each charged
particle will migrate in a pattern determined by its particular property due to changes in
speed and direction, allowing for the separation of biomolecule components with similar
properties.

Parts of Gel Electrophoresis Apparatus

1. Power supply
 The conditions for electrophoresis
are constant current, voltage, or
power.
 A steady power supply should be used
to maintain the migration pace.
 Lead cables with the colors red
(anode/ positively charged electrode)
and black (cathode/ negatively
electrode) link the power supply to
the gel box.
 These wires deliver the gel box with
the electric current coming from the
power source.
 If the current increases, more heat
is produced through resistance, which causes the dissolved ions to stir thermally.
 Water from the equipment will evaporate more quickly.
 Ion concentration in the buffer will rise as a result of this.
  Because DNA and RNA are negatively charged, the black wire is attached to the rear
of the box, which allows them to travel to the front of the gel box, where the
positively charged red wire is attached.

2. Buffers
 The buffer establishes the pH of the system and the electrical charge on the solute.
 The ideal buffer has the following properties:
o Preserve the analyte’s ability to dissolve
o Keep the buffering capacity constant throughout the analysis
o It shouldn’t prevent the intended analytes from being detected.
o Achieve the appropriate range of separation
 Two types of buffers exist Acidic buffer and a Basic buffer
o For a lower pH, acidic buffers, including citrate, acetate, formate, and
phosphate, are utilized.
o Basic buffers like tric, borate, and tricine are employed to keep pH levels
high.
 The valency (ionic strength) and molality of the buffers are equal. Hence they are
composed of monovalent ions.
 The prepared buffers should be carefully chilled while not in use since they can act
as a favorable environment for the growth of bacteria.
 It is possible to use the cold buffer in the procedure since it increases sample
resolution and reduces solvent evaporation.
 The buffer can be reused in large volumes up to four times, but in lesser volumes,
it can be thrown away right away.
 Although there is a high risk of damaging heat-labile chemicals due to the high heat
created, the higher ionic strength of the buffer is advantageous in obtaining a
sharper resolution.

3. Support Media
 Supporting media include starch, polyacrylamide, agarose, and the membrane made of
cellulose acetate in the form of sheets, slabs, and columns.
 It is a colloid that contains more than 90% water.
 It serves as a molecular sieve through which molecules are separated.
 Small molecules can pass through it because it is porous, while larger molecules
cannot.
 Electrical neutrality is required.
 Agarose gel is now mostly employed as a support medium while conducting
electrophoresis.
a. Starch Gel
o It is the first gel medium used for electrophoresis.
o It facilitates the separation of proteins based on charge-to-mass ratio and
molecular size.
o A colloidal suspension was prepared by boiling the suspension of starch
granules in a buffer when allowed to cool sets as a semisolid gel due to the
intertwining of the branched chains of amylopectin.
o Petroleum jelly is added to avoid swelling and shrinking.
o Sharp zones and high resolving power can be achieved.
o As gel preparation with reproducibility is challenging, it is not currently
used.
b. Cellulose Acetate
o When Kohn showed how to separate the protein hemoglobin found in red blood
cells and to spot aberrant hemoglobin in blood serum, cellulose acetate
electrophoresis was first developed.
o Filter papers, made entirely of cellulose, are acetylated to produce cellulose
acetate. The glucose ring’s C-3 and C-6 locations are typically where
acetylation occurs. Compared to other common electrophoretic matrices like
agarose and polyacrylamide, cellulose acetate has bigger pores.
c. Agarose
 o Agar isolated from red seaweeds contains agarose, a naturally occurring linear
polymer composed of galactose and 3,6- anhydro-galactose chains.
o Like agar, agarose is kept as a dry powder in storage.
o Agarose gel is cast by dissolving the agarose powder in the appropriate
solution buffer, heating it, and letting it cool to room temperature.
o The agarose concentration in the solution buffer controls the pore size of the
gel.
o To distinguish between DNA and RNA molecules, agarose gel is frequently used
at 0.8% (W/V) to 5% (W/V).
o Relatively poor resolution compared to polyacrylamide gels.
o It has a low gelling temperature, a neutral charge, and forms stable gels.
Thus, it is considered to be the perfect material for gel electrophoresis. It
can either be solid or liquid.
d. Polyacrylamide
o It is a clear, transparent gel formed by the copolymerization of acryl amide
monomers in the presence of the crosslinking agent N, N- methylene- bis-
acrylamide (also known as “bis-acrylamide”).
o Acrylamide concentration, which must be in proportion to its crosslinking
agent, controls the size of the pores in polyacrylamide gels.
o Separating DNA and proteins typically requires a small amount of acrylamide
gel (3%-15%).
o In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE),
proteins are separated under denatured conditions according to their size,
where a higher percentage of acrylamide gel (10%-20%) is typically used.

4. Electrophoresis chamber
 It is a plastic container or tank filled with a buffer to prevent biomolecule
movement.
 Its transparent lid makes it simple to see the migration process.
 It is wired to a power supply.

5. Container for staining and de-staining gel

 Gel staining and de-staining can be accomplished using trays and containers.
 There are lidded boxes and open-form boxes available.
 They typically have a propylene base.
 They are transparent and closely fitted.
 They are resistant to chemicals and stains.

6. Electrodes
 The two platinum electrodes help separate molecules due to their ability to attract
charges with opposite charges.
 Positive ions are bound by an anode, while a cathode binds negative ions.

7. Gel Caster and Comb

 The gel is poured into a gel caster, which contains the gel and is stored inside the
apparatus once it has dissolved in the solvent.
 The wells are placed using a comb to prepare them for sample loading.

Types of Electrophoresis
There are several types of gel electrophoresis, namely:
1. Paper gel electrophoresis
2. Agarose gel electrophoresis
3. Polyacrylamide Gel Electrophoresis (PAGE)
4. Pulse-field gel electrophoresis (PFGE)
5. SDS- PAGE
6. 2D- electrophoresis
7. Immunoelectrophoresis (Rocket Electrophoresis)
8. Difference Gel Electrophoresis (DIGE)
 They are also categorized as native and denaturing, where RNA or proteins are kept
in their native structure while running through the gel in native gel electrophoresis. In
contrast, the RNA or protein are reduced to their linear structure before or during gel
electrophoresis in denaturing gel electrophoresis. This reduction is achieved by the
addition of a reducing agent to the sample, gel, and/ or buffer, which separates the bonds
within the RNA or protein molecule and results in the formation of a secondary structure.

1. Paper Gel Electrophoresis

 used to investigate serum and other
bodily fluids in clinical settings
 Non-transparent and nontoxic
 Convenient to store
 Protein adsorption
 Poor conductivity
 Background staining
 Cellulose’s OH groups attach to
proteins and slow electrophoretic
motions, causing bands to the trail
and the resolution to be poor.

2. Agarose Gel Electrophoresis

 The concentration of agarose determines the resolution of the electrophoresis.

 It is suited for separating DNA fragments ranging from 100 base pairs to 20 kilobase
pairs.
 Additionally, it applies to the electrophoretic separation of proteins.
 When a low concentration of agarose gel is employed, it can be used to separate
amphoteric molecules based on their isoelectric point, known as isoelectric
focusing.

What is Agarose Gel Electrophoresis?

 Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry,
molecular biology, genetics, and clinical chemistry to separate a mixed population
of macromolecules such as DNA , RNA or proteins in a matrix of agarose.
 Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix
by hydrogen-bonding when heated in a buffer and allowed to cool.
  They are the most popular medium for the separation of moderate and large-sized
nucleic acids and have a wide range of separation.

Principle of Agarose Gel Electrophoresis

Gel electrophoresis separates DNA fragments by size in a solid support medium such
as an agarose gel. Sample (DNA) are pipetted into the sample wells, followed by the
application of an electric current which causes the negatively-charged DNA to migrate
(electrophorese) towards the anodal, positive (+ve) end. The rate of migration is
proportional to size: smaller fragments move more quickly and wind up at the bottom of the
gel.

DNA is visualized by including in the gel an intercalating dye, ethidium bromide.

DNA fragments take up the dye as they migrate through the gel. Illumination with
ultraviolet light causes the intercalated dye to fluoresce.

The larger fragments fluoresce more intensely. Although each of the fragments of a
single class of molecule is present in equimolar proportions, the smaller fragments
include less mass of DNA, take up less dye, and therefore fluoresce less intensely. A
“ladder” set of DNA fragments of known size can be run simultaneously and used to
estimate the sizes of the other unknown fragments.

Requirements/ Instrumentation of Agarose Gel Electrophoresis

The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include:

1. An electrophoresis chamber and power supply

2. Gel casting trays, which are available in a variety of sizes and composed of
UVtransparent plastic. The open ends of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis.

3. Sample combs, around which molten medium is poured to form sample wells in the gel.

4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).

5. Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to
“fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.
6. Staining: DNA molecules are easily visualized under an ultraviolet lamp when
electrphoresed in the presence of the extrinsic fluor ethidium bromide. Alternatively,
nucleic acids can be stained after electrophoretic separation by soaking the gel in a
solution of ethidium bromide. When intercalated into doublestranded DNA, fluorescence of
this molecule increases greatly. It is also possible to detect DNA with the extrinsic
fluor 1-anilino 8-naphthalene sulphonate.

7. Transilluminator (an ultraviolet light box), which is used to visualize stained DNA in
gels.

Steps Involved in Agarose Gel Electrophoresis

1. To prepare gel, agarose powder is mixed with electrophoresis buffer to the

desired concentration, and heated in a microwave oven to melt it.

The concentration of Agarose Gel

 The percentage of agarose used depends on the size of fragments to be

resolved.
 The concentration of agarose is referred to as a percentage of agarose
to volume of buffer (w/v), and agarose gels are normally in the range of
0.2% to 3%.
 The lower the concentration of agarose, the faster the DNA fragments
migrate.
 In general, if the aim is to separate large DNA fragments, a low
concentration of agarose should be used, and if the aim is to separate
small DNA fragments, a high concentration of agarose is recommended.

2. Ethidium bromide is added to the gel (final concentration 0.5 ug/ml) to

facilitate visualization of DNA after electrophoresis.

3. After cooling the solution to about 60oC, it is poured into a casting tray
containing a sample comb and allowed to solidify at room temperature.

4. After the gel has solidified, the comb is removed, taking care not to rip
the bottom of the wells.

5. The gel, still in plastic tray, is inserted horizontally into the

electrophoresis chamber and is covered with buffer.
6. Samples containing DNA mixed with loading buffer are then pipetted into
the sample wells, the lid and power leads are placed on the apparatus, and a
current is applied.

7. The current flow can be confirmed by observing bubbles coming off the
electrodes.

8. DNA will migrate towards the positive electrode, which is usually colored
red, in view of its negative charge.

9. The distance DNA has migrated in the gel can be judged by visually
monitoring migration of the tracking dyes like bromophenol blue and xylene
cyanol dyes.

Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis is a routinely used method for separating
proteins, DNA or RNA.

 Estimation of the size of DNA molecules

 Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic
fingerprinting
 Separation of restricted genomic DNA prior to Southern analysis, or of
RNA prior to Northern analysis.
 The agarose gel electrophoresis is widely employed to estimate the size
of DNA fragments after digesting with restriction enzymes, e.g. in
restriction mapping of cloned DNA.
 Agarose gel electrophoresis is commonly used to resolve circular DNA
with different supercoiling topology, and to resolve fragments that
differ due to DNA synthesis.
 In addition to providing an excellent medium for fragment size analyses,
agarose gels allow purification of DNA fragments. Since purification of
DNA fragments size separated in an agarose gel is necessary for a number
molecular techniques such as cloning, it is vital to be able to purify
fragments of interest from the gel.

Advantages of Agarose Gel Electrophoresis

 For most applications, only a single-component agarose is needed and no
polymerization catalysts are required. Therefore, agarose gels are
simple and rapid to prepare.
 The gel is easily poured, does not denature the samples.
 The samples can also be recovered.

Disadvantages of Agarose Gel Electrophoresis

 Gels can melt during electrophoresis.
 The buffer can become exhausted.
 Different forms of genetic material may run in unpredictable forms.

3. Polyacrylamide gel electrophoresis (PAGE)

 It is used at a concentration of up to 3-30% (pH range: 4-9.0): protein separation
requires a higher concentration than DNA separation, and vice-versa.
 A greater degree of reliability and accurate porosity.
  Its application can be seen in calculating DNA’s molecular weight, DNA sequencing,
studying DNA purity, analysis of recombinant DNA molecules and separation of RNA
molecules, and measuring the molecular weight of RNA.

 Electrophoresis through agarose or polyacrylamide gels is a standard method used to

separate, identify and purify biopolymers, since both these gels are porous in
nature.
 Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of
acrylamide with a cross-linking agent, usually N,N’-methylenebisacrylamide.
 The reaction is a free radical polymerization, usually carried out with ammonium
persulfate as the initiator and N,N,N’,N’-tetramethylethylendiamine (TEMED) as the
catalyst.
 Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in
biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to
separate biological macromolecules, usually proteins or nucleic acids, according to
their electrophoretic mobility.
 The most commonly used form of polyacrylamide gel electrophoresis is the Sodium
dodecyl suplhate Polyacrylamide gel electrophoresis (SDS- PAGE) used mostly for the
separation of proteins.

SDS-PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to

separate components of a protein mixture based on their size.

The technique is based upon the principle that a charged molecule will migrate in an
electric field towards an electrode with opposite sign. The general electrophoresis
techniques cannot be used to determine the molecular weight of biological molecules
because the mobility of a substance in the gel depends on both charge and size.

To overcome this, the biological samples needs to be treated so that they acquire
uniform charge, then the electrophoretic mobility depends primarily on size. For this
different protein molecules with different shapes and sizes, needs to be denatured (done
with the aid of SDS) so that the proteins lose their secondary, tertiary or quaternary
structure .The proteins being covered by SDS are negatively charged and when loaded onto a
gel and placed in an electric field, it will migrate towards the anode (positively charged
electrode) are separated by a molecular sieving effect based on size. After the
visualization by a staining (protein-specific) technique, the size of a protein can be
calculated by comparing its migration distance with that of a known molecular weight
ladder (marker).

Requirements for Polyacrylamide Gel Electrophoresis (PAGE)

 Acrylamide solutions (for resolving & stacking gels).
 Isopropanol / distilled water.
 Gel loading buffer.
 Running buffer.
  Staining, destaining solutions.
 Protein samples
 Molecular weight markers.

The equipment and supplies necessary for conducting SDS-PAGE includes:

 An electrophoresis chamber and power supply.
 Glass plates (a short and a top plate).
 Casting frame
 Casting stand
 Combs

Steps Involved in Polyacrylamide Gel Electrophoresis (PAGE)

1. Sample preparation

 Samples may be any material containing proteins or nucleic acids.

 The sample to analyze is optionally mixed with a chemical denaturant if so desired,
usually SDS for proteins or urea for nucleic acids.
 SDS is an anionic detergent that denatures secondary and non–disulfide–linked
tertiary structures, and additionally applies a negative charge to each protein in
proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the
nucleic acid, causing the constituent strands to anneal. Heating the samples to at
least 60 °C further promotes denaturation.
 A tracking dye may be added to the solution. This typically has a higher
electrophoretic mobility than the analytes to allow the experimenter to track the
progress of the solution through the gel during the electrophoretic run.

2. Preparation of polyacrylamide gel

 The gels typically consist of acrylamide, bisacrylamide, the optional denaturant

(SDS or urea), and a buffer with an adjusted pH.
 The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is
generally about 1 part in 35. The acrylamide concentration of the gel can also be
varied, generally in the range from 5% to 25%.
 Lower percentage gels are better for resolving very high molecular weight molecules,
while much higher percentages of acrylamide are needed to resolve smaller proteins,
  Gels are usually polymerized between two glass plates in a gel caster, with a comb
inserted at the top to create the sample wells.
 After the gel is polymerized the comb can be removed and the gel is ready for
electrophoresis.

3. Electrophoresis

 Various buffer systems are used in PAGE depending on the nature of the sample and
the experimental objective.
 The buffers used at the anode and cathode may be the same or different.
 An electric field is applied across the gel, causing the negatively charged proteins
or nucleic acids to migrate across the gel away from the negative and towards the
positive electrode (the anode).
 Depending on their size, each biomolecule moves differently through the gel matrix:
small molecules more easily fit through the pores in the gel, while larger ones have
more difficulty.
 The gel is run usually for a few hours, though this depends on the voltage applied
across the gel.
 After the set amount of time, the biomolecules will have migrated different
distances based on their size.
 Smaller biomolecules travel farther down the gel, while larger ones remain closer to
the point of origin.
 Biomolecules may therefore be separated roughly according to size, which depends
mainly on molecular weight under denaturing conditions, but also depends on higher-
order conformation under native conditions.

4. Detection
 Following electrophoresis, the gel may be stained (for proteins, most commonly with
Coomassie Brilliant Blue or autoradiography; for nucleic acids, ethidium bromide;
or for either, silver stain), allowing visualization of the separated proteins, or
processed further (e.g. Western blot).
 After staining, different species biomolecules appear as distinct bands within the
gel.
 It is common to run molecular weight size marker sof known molecular weight in a
separate lane in the gel to calibrate the gel and determine the approximate
molecular mass of unknown biomolecules by comparing the distance traveled relative
to the marker.

Applications of Polyacrylamide Gel Electrophoresis (PAGE)

 Measuring molecular weight.
 Peptide mapping.
 Estimation of protein size.
 Determination of protein subunits or aggregation structures.
 Estimation of protein purity.
  Protein quantitation.
 Monitoring protein integrity.
 Comparison of the polypeptide composition of different samples.
 Analysis of the number and size of polypeptide subunits.
 Post-electrophoresis applications, such as Western blotting.
 Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic
Acid.
 Pouring and Running a Protein Gel by reusing Commercial Cassettes.
 Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes.
 Detection of Protein Ubiquitination.

Advantages of Polyacrylamide Gel Electrophoresis (PAGE)

 Stable chemically cross-linked gel
 Greater resolving power (Sharp bands)
 Can accommodate larger quantities of DNA without significant loss in resolution
 The DNA recovered from polyacrylamide gels is extremely pure
 The pore size of the polyacrylamide gels can be altered in an easy and controllable
fashion by changing the concentrations of the two monomers.
 Good for separation of low molecular weight fragments

Disadvantages of Polyacrylamide Gel Electrophoresis (PAGE)

 Generally more difficult to prepare and handle, involving a longer time for
preparation than agarose gels.
 Toxic monomers
 Gels are tedious to prepare and often leak
 Need new gel for each experiment Stable chemically cross-linked gel

4. Pulsed Field Gel Electrophoresis (PFGE)

 In 1984, Shwartz and Cantor introduced this method.
 DNA separation in an agarose gel is accomplished by changing the direction and
strength of the electrical field between electrodes.
 High molecular weight DNA with many megabases or even entire chromosomes are
separated using this technique.
 PFGE is employed in many fields because it produces precise results that are
efficiently reproducible.
 It is applied in the studies on the molecular biology of pathogens found in food,
tracking the genetic stability of organisms employed in the fermentation process,
mapping applications such as chromosome rearrangement detection, RFLP, and DNA
fingerprinting, and identifying linked strains in the event of hospital outbreaks,
etc.

 Pulsed Field Gel Electrophoresis (PFGE) is a technique used for the separation of
large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric
field that periodically changes direction.
 As DNA larger than 15-20kb migrating through a gel essentially moves together in a
size-independent manner, the standard gel electrophoresis technique was unable to
separate very large molecules of DNA effectively which led to the practice of pulsed
field gel electrophoresis.
 In 1982, Schwartz introduced the concept that DNA molecules larger than 50 kb can
be separated by using two alternating electric fields.
Principle of Pulsed Field Gel Electrophoresis (PFGE)
While in general small fragments can find their way through the gel matrix more
easily than large DNA fragments, a threshold length exists above 30–50 kb where all large
fragments will run at the same rate, and appear in a gel as a single large diffuse band.

However, with periodic changing of field direction, the various lengths of DNA react
to the change at differing rates. That is, larger pieces of DNA will be slower to realign
their charge when field direction is changed, while smaller pieces will be quicker. Over
the course of time with the consistent changing of directions, each band will begin to
separate more and more even at very large lengths. Thus separation of very large DNA
pieces using PFGE is made possible.

The Method of Pulsed Field Gel Electrophoresis (PFGE)

The procedure for this technique is relatively similar to performing a standard gel
electrophoresis except that instead of constantly running the voltage in one direction,
the voltage is periodically switched among three directions; one that runs through the
central axis of the gel and two that run at an angle of 60 degrees either side.
The pulse times are equal for each direction resulting in a net forward migration of the
DNA.
The major steps involved in Pulsed-field gel electrophoresis are:

1. Lysis: First, the bacterial suspension is loaded into an agarose suspension. This is
done to protect the chromosomal DNA from mechanical damage by immobilizing it into agarose
blocks. Then the bacterial cells are lysed to release the DNA. The agarose-DNA suspension
is also known as plug mold.

2. Digestion of DNA: The bacterial DNA is treated with unusual cutting restriction enzymes
so that it yields less number of larger size DNA fragments (in contrast to frequently used
restriction enzymes used in RFLP which produces large number of smaller fragments).

3. Electrophoresis: The larger pieces of DNA are subjected to pulse field gel
electrophoresis by applying electric current and altering its direction at regular
intervals (in contrast to the conventional agarose gel electrophoresis done to separate
the smaller fragments where the current is applied in a single direction).

4. Analysis: The fragments of different organisms generated by PFGE are compared to

standards manually or by computer software like BioNumerics.

Applications of Pulsed Field Gel Electrophoresis (PFGE)

 Since, field gel electrophoresis allows the separation of DNA fragments containing
up to 100,000 bp (100 kilobase pairs, or kbp), characterization of such large
fragments has allowed construction of a physical map for the chromosomes from
several bacterial species.
 PFGE may be used for genotyping or genetic fingerprinting.
 It is commonly considered a gold standard in epidemiological studies of pathogenic
organisms.
 Subtyping has made it easier to discriminate among strains of Listeria monocytogenes
and thus to link environmental or food isolates with clinical infections.
Advantages of Pulsed Field Gel Electrophoresis (PFGE)
 PFGE separates DNAs from a few kb to over 10 Mb pairs.
 PFGE subtyping has been successfully applied to the subtyping of many pathogenic
bacteria and has high concordance with epidemiological relatedness.
 PFGE has been repeatedly shown to be more discriminating than methods such as
ribotyping or multi- locus sequence typing for many bacteria.
 PFGE in the same basic format can be applied as a universal generic method for
subtyping of bacteria. (Only the choice of the restriction enzyme and conditions for
electrophoresis need to be optimized for each species.)
 DNA restriction patterns generated by PFGE are stable and reproducible.

Limitations of Pulsed Field Gel Electrophoresis (PFGE)

 Time-consuming.
 Requires trained and skilled technicians.
 Does not discriminate between all unrelated isolates.
 Pattern results vary slightly between technicians.
 Can’t optimize separation in every part of the gel at the same time.
 Don’t really know if bands of the same size are the same pieces of DNA.
 Bands are not independent.
 Change in one restriction site can mean more than one band change.
 “Relatedness” should be used as a guide, not true phylogenetic measure.
 Some strains cannot be typed by PFGE.
5. Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE)
 Originally called the Laemmli Method after its British inventor U.K. Laemmli.
 Upper stacking gel has larger pores with a pH of 6.8, and Lower Separating Gel has
smaller pores with a pH of 8.
 Proteins are separated based on polypeptide chain length in SDS-PAGE, which largely
eliminates the influence of the structure and charge thanks to the use of sodium
dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel.
 SDS, a detergent in the sample buffer, and some reducing chemicals work together to
damage the tertiary structure of proteins by rupturing their disulfide links.
  It is used to calculate the protein’s molecular weight and determine whether
protein samples are pure or not.

Isoelectric point and Isoelectric focusing (IEF)

The pH level known as the isoelectric point is the one where proteins have no net
charge (pI). Proteins are separated by their isoelectric points within a continuous pH
gradient using the high-resolution approach known as isoelectric focusing (IEF). Compounds
that differ in pI by only 0.01 pH units can be separated thanks to the excellent resolving
power.

It is used to distinguish isoenzymes, fractionate proteins, and separate all

amphoteric substances.

6. 2D gel electrophoresis
It is used to analyze complicated protein mixtures and was created as a hybrid of
the 2DGel, IEF, and SDS-PAGE procedures.
IEF separates the protein into its charges in the first step and later according to its
mass in the second step.

SDS treatment makes the separated protein on the IEF gel negatively charged, and the
electrophoresis is carried out by placing the gel horizontally inside the SDS-PAGE gel.
As a result, the proteins that are concentrated on the pI are divided based on their
molecular weights.

7. Immuno-electrophoresis (Rocket Electrophoresis)

In the process of immune-electrophoresis (IEP), firstly, electrophoresis is used to
separate the protein antigen in semi-solid media, and then an immunodiffusion against the
antiserum results in the creation of precipitin.

Suitable antibodies complementary to the test antigen to be measured are dissolved

in molten agar solution and placed on a horizontal plate. Antigens are injected into wells
drilled in the gel. Ag picks up a negative charge at the alkaline pH, moves in the anode
direction, interacts with Ab to form the Ag-Ab complex, ad precipitates. The immuno-
precipitates will then appear as arcs like rockets once the gel has been stained with a
suitable dye like CBB.

Rocket Immunoelectrophoresis- Objectives, Principle, Procedure, Results, Uses

 Rocket Immunoelectrophoresis is an adaptation of radial immunodiffusion developed by

Laurell. It is also known as electroimmunoassay or electroimmunodiffusion.
 It is called as “rocket electrophoresis” due to the appearance of the precipitin
bands in the shape of cone-like structures (rocket appearance) at the end of the
reaction.
 In rocket immunoelectrophoresis, antigen migrates in an electric field in a layer of
agarose containing an appropriate antibody.
 The migration of the antigen toward the anode gives rise to rocket-shaped patterns
of precipitation. The area under the rocket is proportional to antigen
concentration.

Objectives of Rocket Immunoelectrophoresis

 To detect antigen-antibody complexes.
 Determine the concentration of antigen in an unknown sample.

Principle of Rocket Immunoelectrophoresis

Rocket immunoelectrophoresis is a quantitative one-dimensional single electro-

immunodiffusion technique. In this method antibody is incorporated in the gel at a pH
value at which the antibodies remain essentially immobile. Antigen is placed in wells cut
in the gel.
 Electric current is then passed through the gel, which facilitates the migration of
negatively charged antigens into the agar. As the antigen moves out of the well and enters
the agarose gel, it combines with the antibody to form immune complex which becomes
visible.

During the initial phase there is considerable antigen excess over antibody and no
visible precipitation occurs. However, as the antigen sample migrates further through the
agarose gel, more antibody molecules are encountered that interact with the antigen to
form immune complex. This results in formation of a precipitin line that is conical in
shape, resembling a rocket.

The greater the amount of antigen loaded in a well, the further the antigen will
have to travel through the gel before it can interact with sufficient antibody to form a
precipitate. Thus, the height of the rocket, measured from the well to the apex and area
are directly proportional to the amount of antigen in the sample.

Materials Required for Rocket Immunoelectrophoresis

 Agarose
 Antigen
 Antiserum
 Assay Buffer
 Electrophoresis apparatus
 Glass slides

Procedure of Rocket Immunoelectrophoresis

1. About 15 ml of 1 % agarose gel is prepared.
2. The solution is cooled to 55-60oC and 250 µl of antiserum added to 13 ml of agarose
solution. It is well mixed for uniform distribution of antibodies.
3. Agarose solution containing the antiserum is poured onto to grease-free glass plate
placed on a horizontal surface and the gel is allowed to set for 30 minutes.
4. The glass plate is on the template and wells punched with the help of a gel puncher.
5. 10 µl of the standard antigen and test antigen samples are added to the wells.
6. 1X TBE buffer is poured into the electrophoresis tank such that it just covers the
gel.
7. Electrophoresis is carried out at 80-120 volts and 60-70 mA until the antigen
travels 3-4 cms from the well.
8. The glass plate is incubated in a moist chamber overnight at 37o C and the results
interpreted.
9. In case positive for reaction, the tips of the precipitin peaks are marked and the
peak height measured from the upper edge of the well to the tip of the peak.
10. A graph is plotted of the rocket height (on Y-axis) versus the concentration
of antigen (on X-axis) on a semi-log graph sheet. The concentration of the unknown
is determined from the graph by finding the concentration against the rocket height.

Result Interpretation of Rocket Immunoelectrophoresis

  A precipitation ‘rocket’ spreading out from the loading well indicate positive
reaction or specific antigen-antibody reaction due to the presence of antibody
specific to the antigen.
 The absence of the precipitation indicates no reaction or the absence of any
corresponding antibody – antigen.
 The height of the rocket, and its area are directly proportional to the amount of
antigen in the sample, that is, the height of the precipitin peak depends on the
concentration of antigens loaded in the corresponding wells.

Applications of Rocket Immunoelectrophoresis

1. Rocket electrophoresis is used mainly for quantitative estimation of antigen in the
serum.
2. The method has been used for quantization of human serum proteins before automated
methods became available.
3. Determining the concentration of a specific protein in a protein mixture.
4. In estimation of immunoglobulin protease activity.
5. Studies dealing with antigenic relationships between organisms.
6. In enzyme activity electrophoresis.

Advantages of Rocket Immunoelectrophoresis

 Simple, quick, and reproducible method.
 Several unknown samples can be analyzed on a single plate.
 Concentrations of proteins as little as 1 µg/mL can be measured requiring as little
as 20 ng of protein to be loaded in a well.

Limitations of Rocket Immunoelectrophoresis

 These techniques allow quantitative analysis of antigens, but are not applicable to
complex mixtures.

Related Techniques
Fused rocket immunoelectrophoresis
 modification of one-dimensional quantitative immunoelectrophorsis used for detailed
measurement of proteins in fractions from protein separation experiments.

Two-dimensional immunoelectrophoresis
 variant of rocket electrophoresis.
 The test is a two-stage procedure.
 In the first stage, antigens in solution are separated by electrophoresis.
 In the second stage, electrophoresis is carried out again, but perpendicular to that
of first stage to obtain rocket-like precipitation.
9. Difference Gel Electrophoresis (DIGE)
 It is created to address the quantitative element of differential-expression
investigations and to alleviate some of the issues with 2D-PAGE, such as analytical
fluctuations.
 To see each protein sample separately, up to three different protein samples can be
tagged with fluorescent dyes that are size and charge-matched ( for example, Cy3,
Cy5, Cy2). The three samples are combined, loaded, and subjected to 2D
electrophoresis.

Operating procedures of Electrophoresis

Gel solution preparation: A gel is prepared by dissolving it in boiling water. After
cooling to a more comfortable temperature, the solution is poured into a mold or caster.

Gel casting: A comb is used to create wells in the gel once it has been set. The gel is
then inserted into the electrophoretic chamber. Buffer fills the chamber to a maximum of
one-third of its total volume.

Sample preparation: To give the sample color and density, loading dye is added, which can
be either a fluorescent tag or ethidium bromide.

The DNA is isolated and pre-processed, and placed in a solution with some basic blue dye
to help visualize the movement of the sample through the gel.

Sample loading: A clean micropipette is used to load the sample into the wells.

Electrophoresis: The chamber and a power supply where the voltage is set are connected by
the negative and positive leads, respectively. The electric field and negatively charged
particles are created when the power supply is turned on. DNA that is negatively charged
migrates toward the anode because molecules gravitate toward electrodes with opposing
charges.

Stopping electrophoresis, Staining, and Visualization: Dye is used to following the

migration visually. The power supply is turned off. The gel is stained and visualized
using a gel imager when the procedure is finished. By comparing the size of the sample
fragments to the standard, the logarithm of the molecular weight is used to calculate
their sizes.

Applications of Electrophoresis
 DNA fingerprinting to separate DNA fragments to investigate crime scenes and
paternity testing.
 Detection of genetic variations and proteins implicated in health and illness.
 It is employed in the detection and purification of nucleic acids and proteins for
scientific purposes.
 It helps to find pathogens in the blood, other tissues, or sources like food.
 It facilitates the identification and purification of proteins or nucleic acids
frequently examined in greater detail using mass spectrometry or DNA sequencing.
 It is used in blotting methods to analyze macromolecules and evolutionary studies.
 It facilitates the evaluation of results of Polymerase Chain Reaction (PCR).
 Vaccine development and manufacturing both benefit from electrophoresis.
 To differentiate species and evolutionary relationships, taxonomy-DNA profiling is
performed.

Advantages of Electrophoresis
 Reasonably affordable.
 Establishes a direct connection between similar results
 Quite easy to carry out
 Can test DNA from any type of evidence.
 Superior resolution
 Available in a wide range of pore sizes.
  Stable over a wide range of pH, temperature, and ionic strength
 Transparent to light
 Chemically inert
 Electric neutrality and hydrophilicity

Limitations of Electrophoresis
1. Limited sample analysis
 Gene expression can be examined at each little location of a tissue sample using
methods like in situ hybridization (ISH).
 With ISH, researchers may examine every brain region in a sample, whereas
electrophoresis methods can only do so for a limited number of regions.
2. Measurements are not precise
 Gel electrophoresis can efficiently separate proteins with similar molecular weights
using Western blotting.
 It can also separate proteins more precisely using a method called 2D
electrophoresis.
 Mass spectroscopy must be used after the protein has been purified to determine the
precise mass of proteins.
3. A substantial starting sample required
 Amplification of proteins is impracticable as done for DNA and RNA before
electrophoresis. Thus, a sizable tissue sample is required to run these assays,
which reduces the technique’s utility, and flow cytometry and immunohistochemistry
are frequently used to analyze the protein expression in individual cells.
4. Limited visualization facility
 Electrophoresis is ineffective for measuring small hormones, neurotransmitters, and
ions.
 Due to two issues, they don’t fully react to the electrophoresis preparation
(commonly referred to as SDS-PAGE), and even if they did, they are too tiny to
separate properly. They would rush out of the gel’s bottom.
5. Low throughput
 Low throughput in the sense that it doesn’t generate data very quickly. Compared to
PCR and flow cytometry, which are massively parallel and serial processes,
electrophoresis is inferior at producing research data and creating intricate
relationships.

Precautions
 It is advised to use nonconducting floors and benches (made of wood or plastics).
 Avoid unintended grounding points and conductors (such as sinks and other waste
sources) when operating around or close to an electrophoresis system.
 Avoid pushing hard while loading samples, as it may destroy wells.
 Put on gloves, face masks, and goggles while preparing gel.
 EtBr is carcinogenic, and mutagenic therefore take appropriate precautions before
handling it.

Examples of Electrophoresis System

1. DNA electrophoresis system GEP-TH-1000TBT (Manufacturer: Bioevopeak)
Features:
 The system features a built-in high-current power source that can quickly achieve
high efficiency and rapid transfer by directly controlling the current between the
titanium anode and the stainless-steel cathode.
 The transfer system seamlessly incorporates conventional transfer technologies,
allowing for the quickest and most efficient protein transfer from gel to membrane.
2. Electrofocusing electrophoresis system BT105 (Manufacturer: G BIOSCIENCES)
Features:
 Removes gel leak issues; no taping needed.
 There are two combs available for small and large wells.
 Supplied with a selectable power source.
 Easy to use and lightweight.

3. DNA electrophoresis system EPS-2014 (Manufacturer: INOVIALAB)

Features:
  The EPS-2014 Mini Electrophoresis System is a compact, and clever design
specifically for DNA and RNA electrophoresis.
 Due to a magnetic sensor, current can only flow to the electrodes while the lid is
open.
 When the lid is removed or opened while the system is operating, the current is
promptly cut off.

4. Isoelectric focusing electrophoresis system SymphonyIEF (Manufacturer: Hercuvan)

Features:
 A versatile machine called SymphonyIEF Isoelectric Focusing can handle most IEF
needs, from small-scale to high-throughput operations.
 It works with IEF and PAGE horizontal precast gels when the electrode frame is
attached directly to the cooling plate.
DNA Ladders (1 kb, 1 kb plus, 100 bp, 100 bp plus) and Uses

DNA ladder is a solution of DNA molecules of different lengths used in agarose or

acrylamide gel electrophoresis which is used as a reference to estimate the size of
unknown DNA molecules separated on the basis of their mobility in an electrical field
through the gel. DNA ladders are essential molecules routinely used in every DNA dealing
laboratory.

 DNA ladders are also called molecular-weight size markers as they help to
distinguish different DNA fragments based on their molecular weight, which in turn
distinguishes them in terms of size.
 They have been used in various DNA related procedures ranging from the distinction
between DNA variants to quantifying the number of mutations in a DNA fragment.
 The DNA ladder stains well with nuclear stains like ethidium bromide, which allows
the visualization of DNA fragments after gel electrophoresis.
 Commercially available DNA ladders come in the 50 bp, 100 bp, 1000 bp, and 3000 bp
form.
 These ladders are created by the digestion of known-length DNA fragments from
natural sources by the restriction enzymes.
 The length of the fragments, thus, dependent on the restriction enzyme being used,
this makes the process not entirely controllable.
 To overcome this disadvantage and make the ladder more flexible, DNA engineering was
developed.
 Thus, for commercial purposes, a DNA fragment that contains a tandem repeat unit
separated by the same unique restriction enzyme sites was cloned into a plasmid and
then partially digested to produce a ladder with multimers of the repeats.
 More recently, however, many laboratory protocols describing the preparation of DNA
ladders by employing the polymerase chain reaction (PCR) method have been reported.
 This method involves either the simultaneous amplification of a DNA target using
primer sets or the separate amplification of different DNA targets using specific
primers.

The DNA ladder being used for electrophoresis should have the following characteristics:
1. The fragments within the ladder should be separable from each other.
2. The concentration of individual fragments should be enough to be visualized after
electrophoresis.
3. The ladder containing loading dye should not affect the specificity of the DNA
ladder.
4. The fragments in the ladder should be stable enough to use for a long time.
5. The ladder should be highly purified, avoiding unnecessary and unknown fragments.
Types of DNA ladder

DNA ladders are of different types depending on the length of the DNA fragments to be
identified or the number of fragments present in the ladder. Some of the common types of
DNA ladder are:

a. 1 kb DNA ladder
 1 kb DNA ladder consists of 13 linear double-stranded DNA fragments which can be
used to determine the size of DNA fragments with 250 bp to 10,000 bp.
 The 1 kb DNA ladder is a unique combination of a number of plasmids digested with
restriction enzymes and PCR products to yield 13 DNA fragments that are suitable for
use as a molecular weight standard for electrophoresis.
 The use of high intensity 1 kb DNA ladder helps in the quick and easy determination
of electrophoresis results.

Types of DNA ladder

DNA ladders are of different types depending on the length of the DNA fragments to
be identified or the number of fragments present in the ladder. Some of the common types
of DNA ladder are:

a. 1 kb DNA ladder
 1 kb DNA ladder consists of 13 linear double-stranded DNA fragments which can be
used to determine the size of DNA fragments with 250 bp to 10,000 bp.
 The 1 kb DNA ladder is a unique combination of a number of plasmids digested with
restriction enzymes and PCR products to yield 13 DNA fragments that are suitable for
use as a molecular weight standard for electrophoresis.
 The use of high intensity 1 kb DNA ladder helps in the quick and easy determination
of electrophoresis results.

Description
 These ladders have reference bands at 1000 bp and 3000 bp for easy orientation.
 1 kb ladder can be bought commercially at various concentrations, but the
recommended load for an electrophoresis run is 0.5 µg (5µl).
 These can be used in either agarose or in polyacrylamide gels with the concentration
of gel at 0.75% to 1%.
 These ladders come with different tracking dyes like bromophenol blue, xylene cyanol
FF.
 The commercially available ladder is diluted to a 1:4 solution in water for use (3
parts water for 1 part ladder).
 75 µl of water is combined with 25 µl of the DNA ladder to make a 100 µl solution.
 Then, 20 µl of the loading dye present with the kit is added, and the solution is
split into 60 µl solutions (0.5 ml microcentrifuge tubes) and stored at -20°C.
 3 µl of this diluted ladder is used per lane for a typical small (40 ml) agarose
gel, which results in a concentration of approximately 0.63 µg of ladder DNA per
lane.
 The 1 Kb ladders can be stored for varying times at varying temperatures like at
25ºC for six months, at 4ºC for 12 months and at -20ºC for 24 months in storage
buffers like 10 mM Tris-HCl or 1 mM EDTA.

Uses of 1 kb DNA ladder

 The most essential and prominent use of the 1 kb DNA ladder is for the determination
of the size of double-stranded DNA fragments of the length of 250 bps to 10,000 bps.
 The determination of the size of DNA fragments allows the quantification of DNA
fragments and their relative size to one another.
 These DNA size standards can be used as controls during the electrophoresis process.

b. 1 kb plus DNA ladder

  1 kb plus DNA ladder is a DNA size standard consisting of DNA fragments of length
ranging from 0.5 kb to 10 kb used for the determination of the size of double-
stranded DNA fragments of length ranging from 250 bp to 25,000 bp.
 1 kb plus DNA ladder consists of 14 individual chromatography-purified DNA
fragments.
 The purified DNA fragments result in sharp and clear bands that help with the
identification process.

Description
 1 kb plus DNA ladders usually have about 14 purified DNA fragments with a reference
band at 1000 and 3000 bp for easy orientation.
 Most 1 kb plus DNA ladders are provided with loading dye which allows a convenient
detection of sharp bands after electrophoresis.
 The amount of DNA or the number of DNA fragments is exact and precise in these
ladders.
 1 kb plus DNA ladders can be used in 0.7 to 1% agarose or polyacrylamide gels.
Uses of 1 kb plus DNA ladder
 The most important use of 1 kb plus DNA ladders is for the determination of the size
of double-stranded DNA in the range of 250 bp to 25,000 bp.
 It helps to determine the size of DNA fragments that are much longer than the usual
fragments, which allows the quantification of DNA fragments and their relative size
to one another.

c. 100 bp DNA ladder

 A 100 bp DNA ladder is a DNA size standard used for the sizing and quantification of
double-stranded DNA of the size in the range of 100 bp to 1500 bp.
 These ladders consist of about 11 highly purified DNA fragments that form separate
clear bands for the identification of other DNA fragments within that range.
Description
 These ladders have reference bands at 500 bp and 1500 bp for easy orientation.
 The ladder is designed with a uniform intensity of DNA bands of different sizes for
a clear view of each band.
 An exact amount of DNA is used in each band that allows approximate quantification
of DNA samples.
 Some of these ladders can even be radio-labeled with T4 polynucleotide kinase or T4
DNA polymerase.
 This double-stranded DNA ladder can be visualized on 1–2% agarose gels after
staining.

Uses of 100 bp DNA ladder

 These ladders can be used for the determination of the size of double-stranded DNA
fragments of the range 100 bp to 1500 bp.
 It can also be used in diagnostic purposes like the molecular detection of pathogens
and characterization of the genetic variability.
 Besides, it can be used for the molecular tagging of disease resistance genes.

d. 100 bp plus DNA ladder

 A 100 bp plus DNA ladder is a DNA size standard used for the sizing and
quantification of double-stranded DNA of the range of 100 bp to 3,000 bp on agarose
or polyacrylamide gels.
 The ladder has about 12 purified DNA fragments that form separate clear bands for
the identification of other DNA fragments within that range.
Description
 These ladders have reference bands at 500 bp and 1500 bp for easy orientation.
 This double-stranded ladder can be used for the visualization in 1–2% gels.
 A defined amount of DNA in each band within the ladder enables approximate
quantification of sample DNA.
 The ladders are generated from PCR and restriction enzyme digestion of double-
stranded DNA.
 The ladder comes with a loading dye and loading buffer to enable the movement of the
fragments through the gel slab.

Uses of 100 bp plus DNA ladder

 A 100 bp plus DNA ladder can be used as a DNA size standard for the visualization of
linear double-standard DNA fragments of the size 100 bp up to 3,000 bp.
 It enables the identification and separation of samples with DNA fragments based on
their molecular weight and size.
 This ladder, like others, can be used for diagnostic purposes for the molecular
detection of various pathogens in different samples.