3. INTRODUCTION
Electrophoresis:- Electrophoresis is a process of migration of charged particle through a
solution under the influence of external electric field.
Gel electrophoresis:- It is the method used for separation and analysis of macromolecules
like DNA, RNA and proteins or their fragments based on their size and charge.
It uses a gel as an anti-convective medium and/or sieving medium during electrophoresis.
An illustration of gel electrophoresis workflow
4. PRINCIPLE
By placing the substance to be separated in wells of the gel and applying an electric
current, allows the molecules to move through the gel matrix at different rates
towards the anode if negatively charged or toward the cathode if positively charged.
Smaller molecules migrate through the gel more quickly and therefore travel further
than larger fragments that migrate more slowly and therefore will travel a shorter
distance.
This results in a separation by size, with the larger molecules nearer the well and the
smaller molecules farther away.
5. TYPES OF GEL ELECTROPHORESIS
Agarose gel electrophoresis
Polyacrylamide gel electrophoresis
Starch gel electrophoresis
6. AGAROSE GEL ELECTROPHORESIS
A method used in biochemistry and molecular biology to separate DNA or RNA
molecules by size.
Most agarose gels are made between 0.7% and 3%.
Low percentage gels are very fragile and are used to separate larger molecules.
High percentage gels are used to separate smaller molecules.
The higher the voltage, the faster the DNA moves.
But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High
voltages also decrease the resolution.
8. POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS PAGE)
Polyacrylamide gel electrophoresis (PAGE) is used for the separation of proteins.
In this method polyacrylamide gel is used as a sieving/filtering material.
Sodium dodecyl sulfate (SDS )is a detergent with a strong protein-denaturing effect and
binds to the protein backbone at a constant molar ratio.
In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for
proper folding, proteins unfold into linear chains with negative charge proportional to
the polypeptide chain length.
The negatively charged SDS molecules repel each other, which lends a rod-like shape to
the SDS-treated proteins.
9.
10. STARCH GEL ELECTROPHORESIS
Partially hydrolysed potato starch used as another non-toxic medium for protein
electrophoresis.
In this method, a suspension of granular starch should be boiled in a appropriate buffer to
give a colloidal suspension.
The suspension on cooling sets as a semisolid gel due to interwining of the branched
chains of amylopectin.
In order to avoid swelling and shrinking petroleum jelly is used.
The gels are slightly more opaque than acrylamide or agarose gel
11. Blotting
techniques
Southern
Blotting
To detect DNA
Northern
Blotting
To detect RNA
Western
Blotting
To detect protein
TYPES OF BLOTTING TECHNIQUES
Blotting:- Blots are techniques for transferring DNA, RNA and proteins onto a
carrier(blotting membrane) from agarose or polyacrylamide gel.
12. SOUTHERN BLOTTING
The technique was developed by E.M. Southern in 1975.
Used to detect the presence of a particular fragment of DNA in a sample.
The DNA detected can be:
- Single gene
- Part of a larger piece of DNA such as viral genome
The key in this method is Hybridization.
Hybridization means the process of forming a double-stranded DNA molecule between a
single-stranded DNA probe and a single-stranded target patient DNA.
13. Steps involved in southern blotting
Genomic DNA
Restriction endonuclease
DNA fragments
Gel electrophoresis
Long DNA fragments and short DNA
fragments on Agarose gel
Denatured by mild alkali blot
transfer
Nitrocellulose or Nylon membrane
(Baked in oven at 80ºC for 2-3 Hrs)
DNA probe
Hybridized bands on
Autoradiograph
Hybridization
Washing of
unbound probe
14. APPLICATIONS
Southern blots are used in gene discovery, mapping, evolution and development
studies, diagnostics & forensics.
Used in diagnosis of disease caused by genetic defects.
Used to identify infectious agents.
To identify mutation or gene rearrangement in the sequence of DNA.
Used for paternity testing, criminal identification & victim identification.
15. NORTHERN BLOTTING
Northern blotting was developed by James Alwine and George Stark in 1979.
This technique used for detection of specific RNA sequences.
RNA is more susceptible to degradation than DNA.
RNA molecules have defined length & much shorter than genomic DNA it is not
necessary to cleave RNA before electrophoresis.
RNA sample are separated based on size by gel electrophoresis.
16. Steps in Northern blotting
RNA isolation
Separation of RNAs using
Gel electrophoresis
Blotting on nitrocellulose membrane
Hybridisation with labelled probe
Washing to remove unbound probe
Detection by autoradiogram
17. APPLICATIONS
Used for studying and inspecting gene manifestation design between tissues, organs,
developmental stages, pathogen infection and over the course of treatment.
Diagnosis of several diseases including viral infections..
Study of RNA degradation and splicing.
To detect specific mRNA molecular weights and contents in a sample.
Study of RNA half-life.
18. WESTERN BLOTTING
Western blotting was developed by W. Neal Burnette in 1981.
It is a technique used for identification of particular protein from the mixture of
protein.
Western blotting is also known as immunoblotting because it uses antibodies to detect
the protein.
Western blotting is based on building antibody – protein complex through specific
binding of antibodies to proteins immobilized on a membrane.
Then detecting the bound antibody through several detection methods.
19. Steps involved in western blotting
a) Extraction of protein: By mechanical or chemical lysis of cell.
b) Gel electrophoresis: Proteins are separated by Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE).
c) Blotting: - Proteins are transferred from the gel to solid support membrane such as
nitrocellulose or polyvinylidene difluoride (PVDF).
- Electro-blotting is mainly used which uses electric field to pull proteins from
the gel onto the PVDF or nitrocellulose membrane.
20. d) Blocking: Antibodies are also proteins they are likely to bind the nitrocellulose paper.
So before adding the primary antibody the membrane is masked by using
casein or Bovine serum albumin (BSA).
CONT….
21. CONT….
e) Treatment with primary antibody: The 1º Ab is specific to desired protein and it
forms Ag-Ab complex.
f) Treatment with secondary antibody: - Secondary antibody is enzyme labelled.
Alkaline phosphate (AP) and Horseradish peroxide (HRP) are the most commonly used
enzymes for protein detection.
- 2º Ab is antibody attach with 1º Ab and forms protein- 1ºAb-2ºAb complex.
22. g) Treatment with suitable substrate: - To visualize the enzyme action, the reaction mixture
is incubated with specific substrate.
- The enzyme convert the substrate to give visible coloured product, so band of colour can
be visualized in the membrane.
- Then detection can be done by several detection methods like colorimetric or
chemiluminescent or radioactive detection.
CONT….
23. APPLICATIONS
To determine the size and amount of protein in given sample.
Useful to detect defective proteins.
Confirmatory test for Hepatitis-B involves western blotting technique.
Diagnosis of HIV by ELISA involves the western blotting technique.
This technique is also employed in the gene expression studies.
24. REFERENCES
Gordon AH. Electrophoresis of proteins in polyacrylamide and starch gels.
Laboratory techniques in biochemistry and molecular biology. 1969.
https://www.researchgate.net/publication/224829868_Gel-
Electrophoresis_and_Its_Applications
Hayes PC, Wolf CR, Hayes JD. Blotting techniques for the study of DNA, RNA,
and proteins. BMJ: British Medical Journal. 1989 Oct 10;299(6705):965.
https://www.biotechnologynotes.com/electrophoresis/electrophoresis-meaning-
definition-and-classification-withdiagram/293
