“Gel Electerophoresis Techniques”

Name : Huma Aslam

Class : B.Sc (H)
Submitted to : Miss. Sumaira Ashraf
Roll number : 629-BH-MB-18
Course : Cell Biology
Course code : MICBIO-2202
Department : Institute of Biotechnology

GCU, LAHORE
Table of context:

Introduction……………………………………………………………………….1

Apparatus and principle of Gel Electrophoresis……………………….…………2

Different types of Gel……………………………………………………………..3

 Agarose
 Polyacrylamides
 Starch
Preparation of electerophoresis on agarose gel- agarose gel
Preparation of Polyacrylamide gel-PAGE
Buffers……………………………………………………………………………4

Down streaming…………………………………………………………………..6

Applications………………………………………………………………………6
 Nucleic Acids
 Proteins
 Nano particles
Conclusion……………………………………….……………………………….10
Introduction:
Gel electrophoresis is a technique to isolate the molecules like DNA, RNA or proteins. It is
on the basis of their size or charge, respectively. As in word electrophoresis, which is an
electromotive force (EMF), actually, the suffix ‘phoresis ’means ‘movement or migration’
while the prefix ‘electro’ accounts that molecules can be separated in the presence of current
or moving charges, whereas, the gel denotes to the ’matrix it contains’. So, electric current
is given in this technique.
It concept was came from "Tiselius apparatus" in 1937 when new chemical and separation
processes were based on this electrophoresis, being developed in 19th century and so
on.iThen, by 1940’s, 1950’s (zone electrophoresis) and 1960’s, it was possible to do gel
electrophoresis, giving rise -in molecular biology.[ CITATION Tis37 \l 1033 ]
It consist of gel, electrophoretic chamber with positive (anode) and negative (cathode) ends
with a supply of electric power by which DNA, RNA or proteins or even, nanoparticles are
move towards specific end (called as sieving).[ CITATION Sam01 \l 1033 ] The gel which is
used in whole scenario, is colloid in a solid form (containing 99% water), to lessen the
thermal convection and serving medium, etc., is covered with buffer.
Thus, on the basis of need, we commonly, divide them
as Agarose gel Electrophoresis, PAG (Polyacrylamide;
the most common one) and Starch Gel Electrophoresis,
discussed in detail later. It can be done by horizontally or
vertically ; Horizontal and Verticle Gel Electerophoresis.
It is used in wide range of field like in clinical chemistry,
in biochemistry (to separate proteins), forensic molecular
biologists and microbiology, etc. This is so because the
results which we devised from this can be prompted with
various other techniques for the required purpose.
Figure a: Showing real-based
gel electerophoresis machine
DNA gel electrophoresis is generally performed for
analytical purposes, often after DNA amplification through the polymerase chain reaction
(PCR), but can be used as a preparatory technique before the usage of other methods such as
mass spectrometry, Polymerise Chain Reaction, cloning, DNA sequencing or Southern blot
for further characterization. Hence, give wide range of applications.

Apparatus and principle of Gel Electerophoresis:

 These techniques are applied in an apparatus. It is, generally, consist of:
 Gel (mostly a cross-linked polymer whose composition is selected on the basis of the need
and material to be analyzed),

 Electrophoretic chamber (which is hard plastic box with opposite charges at both ends
supplied with power supply)
 Buffer (which gives ions and important in the passage of current)

Figure b: Showing Gel Electerophoresis overviewwith its result

However,
the main principle is that when the wells are made on the cathode side and the sample is
loaded, it is placed inside the chamber, which is connected to the power supply, and the
buffer solution is added as needed, as described. When the power is on, the electric or EMF
field is applied to the buffer.
This means that positively charged molecules move towards the anode. (DNA and RNA
have a negative charge, since; however, proteins must be treated with a detergent to impose
a negative charge on them) (Kara Rogers, 2017), in the presence of the electric field E. L It
is important that the more molecules the lighters move faster and on the contrary for the
heavier ones. It also depends on the density and concentrations of the gel for how far the
molecules will travel from the wells of the gel, that is: the speed of program, ν. As, shown in
following equation:
Eq
v=
fv

(Where f is the friction coefficient and q is the net charge on the molecule). [ CITATION
Ada97 \l 1033 ] It is important to note that sometimes stained molecule markers are added
 along with this whole scenario, whose composition and size are known, to oblige as
references. It also allows the conception of the marker while moving through the gel. Gel
coloring - Display: After completing the whole process, visualization can be performed by
staining. Different stains can be used for different molecules. Like, DNA can be visualized
using ethidium bromide by passing through UV light. On the other hand, the protein can be
viewed using the satin silver blue dye or Coomassie. [ CITATION Kar17 \l 1033 ]
Gel-Types:
The range to which DNA or plasmids (circular DNA) travel depends on size or whether this
ulaxed or not , for plasmids.So, usually NaOH of formamide are used to distrupt the
structure. In other case they wouldn’t travel much longer and folds into compliex shapes.

Figure c: Showing overview of DNA gel-electerophoresis

Eukaryotic DNA, genome presents in the form bands of 28s and 18s rRNA, the 28s band
being approximately twice as intense as the 18s band, which are distinct.The electrophoresis
of RNA samples can be used to verify the contamination of genomic DNA and also the
degradation of RNA. And from eukaryotic organisms presents various bands of rRNA 28
and 18. RNA which is degraded has bands in an undefined form, has a stained appearance
and the intensity ratio is lower at 2:1.The main purpose for DNA is:
 To acquire for fingerprinting in forensic purposes.
 To acquire for fingerprinting in paternity testing
 To acquire for fingerprinting so that evolutionary relationships can be studied
 To check PCR reaction
 To study genes with a particular disease.

While DNA fragments can be viewed immediately in UV light, protein fragments need to be
labeled with target-specific antibodies, a process known as immunostaining. This reaction
cannot occur on a gel substrate; the proteins must be transferred onto a membrane (mainly
PVDF or nitrocellulose) and then they must be dried with a solution that contains an
antibody that recognizes and binds specifically to the protein of interest.
Proteins:
They have different shapes and structure, we know, due to presence of different amino acids
sequence and different chains. Hence, for studing proteins, it is necessary to denature them.
It can be done using detergent as SDS (sodium dodecyl sulfate), which is added relative to

Figure d: Procedure for Protein Gel Electerophoresis

the size of protein needed to be studied (as 13.4g SDS/Protein).

It impacts negative charge. Thus, the range to which it travels depends upon its size because
after treating it detergent it will act like long rod.
Proteins are usually investigated using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), by native gel electrophoresis, by preparative gel
electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.
 Figure e: Showind Gel Electerophoresis results of DNA (one is stained ladder) and
RNA (stained withRunBlue Prestained Molecular Weight Markers.

Many different proteins have different sizes because of the presence of various amino acids
sequence and different charges.
Characterization through ligand interaction may be performed by electroblotting or by
affinity electrophoresis in agarose or by capillary electrophoresis as for estimation of
binding constants and determination of structural features like glycan content through lectin
binding.
Nano particles:
For gel electrophoresis is to separate or characterize metal nanoparticles or metal oxide
(e.g. Au, Ag, ZnO, SiO2) as regards the size, shape or surface chemistry of the nanoparticles.
The aim is to obtain a more homogeneous sample (for example, a narrower particle size
distribution), which can be used in other products / processes (for example, self-assembly
processes). The nano particles can be separated in a medium in two ways; wherein the mesh
size can be smaller to protein size or equal to protein. On the basis of which, they can be
move on the medium.[ CITATION Bar20 \l 1033 ]
Two-dimensional electrophoresis has recently been developed, in which charge separation
(IEF) and dimensions (SDS-PAGE) are often used together in two-dimensional
electrophoresis, where charge separation is used for the first time, therefore Le Separate
proteins are separated by size, like proteins.
This is a very effective method of identifying a particular protein in a tissue that can contain
thousands of proteins and where there can be only small differences between the control and
treated samples (for example, to search for a protein involved in predation resistance. by
insects on plants).
Conclusion:
Gel electrophoresis allows scientists to visualize the size of DNA segments and helps
sequence DNA lengths. Sequencing is the process by which scientists learn the exact order
of bases in a length of DNA. In gel electrophoresis, negatively charged DNA fragments
travel through the porous gel to the positive end of the tray when a charge is applied to the
gel. However, large fragments do not travel as much as small fragments.
Therefore, the fragments are separated by size. The first "strip" at the top of a lane
represents a collection of fragments of a certain size, the second strip a second collection of
fragments of a smaller size and so on. Furthermore, it is possible to apply different methods
or techniques based on the purpose and basis of the required analysis or the type of analysis,
also as described in detail. In sequencing, gel electrophoresis is used to separate individual
DNA bases, the order of which can be "read" from the gel. Large-scale sequencing is now
facilitated by sequencing machines.
 References:
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Chromatography , 133-147.
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Techniques of Biochemistry and Molecular Biology. Great Britain: Cambridge University
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6. Kara Rogers, G. L. (2017). Gel electerophoresis.
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biochemistry and biotechnology.
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Applications.
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