2. Table of Content
• Introduction
• Definition of Electrophoresis
• Principle of Electrophoresis
• How does it work?
• Electrophoresis types
• Gel electrophoresis procedure
• Applications
• Advantages of Electrophoresis
• Disadvantages of Electrophoresis
• Conclusion
• References
3. Introduction
Suppose you have just done a PCR reaction, making many copies
of a target DNA region. Or perhaps you’ve done some DNA
cloning, trying to "paste" a gene into a circular DNA plasmid.
Now, you want to check and see whether your PCR worked, or
whether your plasmid has the right gene in it. What technique can
you use to visualize (directly observe) the fragments of DNA?
4. Definition
Electrophoresis is a laboratory technique used to separate
DNA, RNA, or protein molecules based on their size and
electrical charge.
An electric current is used to move molecules to be
separated through a gel. Pores in the gel work like a sieve,
allowing smaller molecules to move faster than larger
molecules.
The conditions used during electrophoresis can be
adjusted to separate molecules in a desired size range.
5. Principle of Electrophoresis
Electrophoresis is based on the phenomenon that most
biomolecules exist as electrically-charged particles, possessing
ionizable functional groups. Biomolecules in a solution at a given
pH will exist as either positively or negatively charged ions.
When subjected to an electric field, ionized biomolecules will
migrate at a different pace, depending on the mass and the net
charge of each particle in the solution—negatively-charged
particle, anions, will migrate towards a positively charged
electrode, or cathode, and cations, or positively-charged
particles, will be pulled towards a negatively-charged electrode
called anode.
6. How does it work?
An electric field is applied to molecules and as they are
electrically charged themselves it results in a force acting
upon them.
The greater the charge of the molecule the greater the
force applied by the electrical field and therefore the
further through the support medium the molecule will
move relative to its mass.
Some example applications of electrophoresis include DNA
and RNA analysis as well as protein electrophoresis which is
a medical procedure used to analyse and separate the
molecules found in a fluid sample (most commonly blood
and urine samples).
7. Electrophoresis types
This technique is divided into two types viz slab
electrophoresis and capillary electrophoresis.
Types of Electrophoresis:
Capillary electrophoresis
Gel electrophoresis
Paper electrophoresis
Slab electrophoresis
Zone electrophoresis
Immunoelectrophoresis
Isoelectrofocusing
8. Capillary electrophoresis
Capillary electrophoresis is an analytical technique that separates ions based on their
electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is
dependent upon the charge of the molecule, the viscosity, and the atom's radius.
The rate at which the particle moves is directly proportional to the applied electric field--the greater
the field strength, the faster the mobility. Neutral species are not affected, only ions move with the
electric field. If two ions are the same size, the one with greater charge will move the fastest.
9. Slab electrophoresis
It is primary method used in Clinical chemistry lab. It has ability to Simultaneously separate Several
samples in one run. It uses a rectangular gel Regardless of thickness. Gels are cast on sheets of
Plastic backing. It is useful in separation of isoenzymes, Lipoproteins, Hemoglobin & fragments Of
DNA & RAN
·Slab Gel Electrophoresis is a type of Gel electrophoresis The gel is set or polymerized into a thin
slab between two glass plates.
10. Gel electrophoresis procedure
Below we have explained the steps conducted during DNA electrophoresis.
Step 1: Prepare sample –
Isolate the DNA and prepare the solution by adding blue dye so that it
will be easy to observe the movement of the sample taking place in the gel.
Step 2: Prepare an agarose TAE gel solution –
TAE buffer solution helps to generate an electric field during the process
of electrophoresis. To prepare the solution, for example, if there is a
requirement of 1% agarose gel then add 100mL TAE to 1 g of agarose. The
higher percentage of agarose will give a denser screen. Dissolve the agarose
by heating the agarose TAE solution.
11. Gel electrophoresis procedure Contt….
Step 3: Gel casting –
Pour the agarose TAE solution in a casting tray. Allow it to cool and solidify. A gel slab along with
the wells is ready to use for the experiment.
Step 4: How to set up the electrophoresis chamber?
Fill a chamber with TAE buffer. Place the solid gel in the chamber. Place the gel in such a position
such that it is near the negative electrode.
Step 5: Gel loading –
Load the wells with the DNA sample and DNA ladder (a reference for sizes).
12. Gel electrophoresis procedure Contt….
Step 6: Process of electrophoresis –
Connect the positive and negative points to the power supply and chamber. Switch on the power
and migration in the DNA sample due to the electric field generated. The negatively charged sample
will move towards the positive point and away from the negative electrode.
Step 7: Observe the DNA –
Once you see the migration of the blue colored DNA samples in the gel switch off the power
supply. Remove the gel and place it in the ethidium bromide solution.
Step 8: Expose the ethidium bromide stained gel under UV light and take a picture. DNA bands
appear in the lane of respective well. Also, the DNA ladder is visible. Therefore, the length of DNA
bands can be determined. Below is the image of the experiment conducted.
13. Applications for Electrophoresis
Antibiotic testing: Antibiotics help medical patients fight off illnesses and
infections. Today, antibiotics are widely used for medical applications, but
they must be researched extensively to ensure their safety, purity and
effectiveness. Electrophoresis is used to separate the antibodies in the
antibiotic from any impurities. This process also enables researchers to
determine the concentration of the antibiotic, making dosage more
accurate.
DNA analysis: DNA analysis is one of the most common applications for
electrophoresis. Using gel as a medium, researchers can stratify DNA into
segments using an electrical charge and keep the molecules in place once
the charge is removed. This allows researchers to examine molecules at high
resolutions, making it much easier to thoroughly analyze DNA structures.
14. Applications for Electrophoresis Contt…
Vaccine testing: Vaccines have saved countless lives and radically reduced
the spread of diseases like measles and pertussis. Electrophoresis has
played an essential role in the development of modern vaccines; it is used
to test the purity and concentration of vaccines. Researchers use
electrophoresis to test variations of vaccines with different levels and types
of antibodies to conduct studies to find the best possible version of a
single vaccine.
Analysis of proteins and antibodies: Another key application for
electrophoresis is protein and antibodies analysis. Electrophoresis that’s
used for this application is called immunoelectrophoretic, and it allows
research to study the interactions between proteins and antibodies.
15. Advantages
(a) High separation efficiency –
(b) Short analysis time –
(c) Low sample and electrolyte consumption –
(d) Low waste generation –
(e) Ease of operation
16. Disadvantages
Electrophoresis Has Limited Sample Analysis
Electrophoresis Measurements Are Not Precise
Substantial Starting Sample is Required
Only Certain Molecules Can Be Visualized
Electrophoresis is Low Throughput
17. CONCLUSION
Although the existence of some pitfalls—such as contamination
or species conversion—cannot be ig-nored, gel electrophoresis is
a powerful tool for the fractionation or separation of high-
molecular-masscompounds, such as metalloproteins.
The two main separation modes, one- or two-dimensional, offer
areal separation of the proteins. While non-denaturing 2DE still
has to be further developed, denaturing2DE is already a very
powerful separation technique for covalently bound elements,
such as Se or evenphosphorus and sulfur.
