This document provides an overview of various blotting techniques used to detect specific biomolecules, including Southern blotting, Western blotting, Northern blotting, and related methods. It describes the basic principles of blotting, which involve transferring biomolecules from a gel to a membrane for detection using probes or antibodies. Examples of applications for each technique are also outlined.

1. BLOTTING TECHNIQUES
Presented by: Ashwini Patil
M. Pharm (Pharmacology) Sem: II
Department of Pharmacology
R.C Patel Institute of Pharmaceutical Education and
Research, Shirpur

2.  Southern blotting is named after its inventor, the
British biologist Edwin Southern (1975)
 Other blotting methods Western (WB), Northern
(NB) that employs similar principles, but using
probes or RNA, have later been named in
reference to Edwin Southern name
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3.  Visualisation of specific DNA ,RNA & Protein
among many thousands of contaminating
molecules requires no. of techniques which are
collectively termed BLOT transfer and the
process is called as Blotting.
 A blot, in molecular biology and genetics, is a
method of transferring proteins, DNA or RNA,
onto a carrier
 (for example, a nitrocellulose, polyvinylidene
fluoride (PVDF) or nylon membrane)
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4. Southern blotting
Western
blotting
Northern
blotting
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1)Far western blot
2)Far eastern blot
3)Reverse northern blot
4)Dot blot

5.  PRINCIPLE:-
 The key to this method is Hybridization.
 Hybridization:-It is a process of forming a double
stranded DNA molecule between a single stranded
DNA probe and single stranded target DNA.
 There are two important features of Hybridization
i.e
1)The reactions are specific-the probes will bind to
targets with complementary sequence.
2)The probe can find one molecule of target in a
mixture of millions of related but non complementary
molecules.
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6. 5

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9.  To identify specific DNA in a DNA sample.
 To isolate desired DNA for construction of DNA.
 To identify mutations, deletions, and gene
rearrangements.
 Used in prognosis of cancer and in prenatal
diagnosis of genetic disease.
 Used in phylogenetic analysis.
 Diagnosis of HIV-1 and infectious disease.
 In DNA fingerprinting
1) Paternity and maternity testing
2) Criminal identification and forensics
3)Personal identification
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10.  Northern blotting is a technique for detection of
specific RNA sequences. Northern blotting was
developed by James Alwine and George Stark at
Stanford University (1979) and was named such by
analogy to Southern blotting.
 PRINCIPLE:-
1)Hybridization
2) Electrophoresis
3) Capillary action
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11. Extract and purify mRNA from cells.
Separated by gel electrophoresis
Transfer to aminobenzyloxymethyl filter
paper.(BLOTTING)
Add labelled DNA probe for hybridization to
take place
Wash off unbound probe
autoradiograph to detect mRNA- DNA hybrid.
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13.  Detecting a specific mRNA in a sample.
 Used in the screening of recombinants by detecting
the mRNA produced by transgenesis.
 In disease diagnosis.
 In gene expression studies.
 mRNA splicing studies.
 Study RNA degradation.
 Study RNA half life.
 Time consuming procedure.
 RNA samples can be degraded by RNAases.
 Use of radioactive probes.
 Detection of multiple probe is a problem.
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14.  The reverse northern blot is a method by
which gene expression patterns may be
analyzed by comparing isolated RNA
molecules from a tester sample to samples in a
control cDNA library.
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15.  Quantification of mRNA expression levels
 Confirmation of differential display results
 DNA Microarrays.
Research Applications:-
1)Reverse northern blotting was used in a 2013 study
in Gene in which the author identified a number of
genes responsible for early cold-resistance response
in the cold-hardy citrus fruit Poncirus trifoliata.
2)A study utilized the technique to determine
differences in striatal tissue in rats treated with 3-NP,
which is often used in experiments to generate a
Huntington’s Disease-like phenotype in rats.
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16. 1
• cDNA sequences for transcripts of interest are
immobilized on nylon membranes
2
• Prepared reverse northern blot membranes are pre-
hybridized in Denhardt’s solution with SSC buffer and
labeled cDNA probes are denatured at 100 °C and added
to the pre-hybridization solution.
3
• The membrane is incubated with the probes for at least
15 hours at 65 °C, then washed and exposed.[3]
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18.  Western blotting uses specific antibodies to
identify proteins that have been separated based
on size by gel electrophoresis. The immunoassay
uses a membrane made of nitrocellulose or
PVDF (polyvinylidene fluoride).
 The gel is placed next to the membrane and
application of an electrical current induces the
proteins to migrate from the gel to the membrane.
 The membrane can then be further processed with
antibodies specific for the target of interest, and
visualized using secondary antibodies and
detection reagents.
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19.  Principle:-
 It is based on the principle of immunochromatography.
 Antigens are separated by Poly Acrylomide Gel
Electrophoresis(PAGE).
 Antibodies in serum react with specific antigens.
 Signals are detected according to the principles of test
systems.
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20.  The technique
uses three
elements
(1) separation by
size
(2) Transfer to a
solid support
(3) Marking target
protein using a
proper primary and
secondary antibody
to visualize.
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21. Thermo Scientific myECL Imager
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22. 21

23.  Used to pinpoint a specific protein in a given
sample, employs the ability of an enzyme or
fluorescence-labeled primary antibody to bind to
its specific antigen.
 Sensitivity:-Its ability to detect as little as 0.1
nanograms of protein in a sample.
 Fewer antibodies are needed for testing, which
cuts down laboratory costs significantly.
 Specificity:-specific antibodies show affinity for
specific proteins, the process can selectively detect
a target protein even in a mixture of 3 lakh
different proteins.
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24.  Prone to False or Subjective Results:- A false-
positive results comes when an antibody reacts
with a non-intended protein, which is what
frequently happens when a patient being tested for
HIV.
 On the other hand false result can easily occur if
larger proteins are not given sufficient time to
transfer properly to the membrane.
 High Cost and Technical Demand:-A delicate
process, western blotting requires precision in
every step for proper identification of a sample's
constituents.
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25.  Highly sensitive method to detect a specific
protein even in very low quantity.
 Used in clinical diagnosis
 Quantifying a gene product (gene expression
studies)
 Some forms of Lyme disease testing employ
Western blotting .
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26. To identify proteins that interact with a given
protein.
Far-Western blot analysis is an alternative method
to analyze protein-protein interactions.
PRINCIPLE:-
The principle of far-western blotting is modified
from western blotting.
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27. 1
• Uses the natively-folded recombinant protein as the first
probe to interact with proteins that have been separated
on a PVDF membrane.
2
• The second probe used in this platform is the specific
antibody against the original protein, while the HRP-
conjugated third probe is the antibody against the second
probe.
3
• When combined with protein identification using LC-
MS-MS, the resulting protein spots (or protein bands) can
be characterized and then subjected to further functional
assays, such as protein immuno-precipitation.
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28.  Low experimental cost.
 High sensitivity.
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29.  Far-Eastern blotting is a technique developed
in 1994 by Taki and colleagues at the Tokyo
Medical and Dental University, Japan for the
analysis of lipids separated by high-
performance thin layer chromatography.
 The lipids are transferred from the HPTLC
plate to a PVDF membrane for further analysis,
for example by enzymatic or ligand binding
assays or mass spectrometry.
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30. 29

31.  Purification of phospholipids.
 Structural analysis of lipids in conjunction with
direct mass spectrometry.
 Binding study using various ligands such as
antibodies, lectins, bacterium, viruses, and
toxins.
 Enzyme reaction on membranes.
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32.  A technique for detecting, analyzing and
identifying proteins, similar to the western blot
technique but differing in that protein samples
are not separated electrophoretically but are
spotted through circular templates directly onto
the membrane or paper substrate.
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33. 32

34.  Estimation of concentration of proteins in
crude preparations (such as culture
supernatant).
 The size of complementary sequences is
obtained through the process.
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35.  http://ourpastimes.com/advantages-
disadvantages-western-blot-8670663.html
 http://www.biologyexams4u.com/2014/01/weste
rn-blotting-principle-summary-of.html
 https://www.researchgate.net/figure/231212350_fi
g1_Figure-1-Schematic-diagram-of-
immunochromatography
 http://www.biologyexams4u.com/2013/12/south
ern-blotting-procedure-steps.html
 http://i-base.info/guides/testing/test-accuracy-
results-and-further-testing
 http://www.aidsmap.com/Confirmatory-
tests/page/1323369/
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36.  http://labtestsonline.org.uk/understanding/a
nalytes/lyme/tab/test/
 http://www.drzeegers.com/2014/08/lyme-
disease-diagnosis-blood-test-tickborne-disease/
 http://www.biologyexams4u.com/2014/01/n
orthern-blotting-principle-summary-of.html
 Sritunyalucksana K, Wannapapho W, Lo CF,
Flegel TW. (2006) PmRab7 is a VP28-binding
protein involved in white spot syndrome virus
infection in shrimp. J Virol 80: 10734-10742
 www.scribd.com
 http://webap.rsh.ncku.edu.tw/shrimpwssv/in
dex.php/sfgc-technological-platforms/protein-
interactions-using-far-western-blotting
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37.  http://www.wikivisually.com/wiki/Reverse_N
orthern_blot
 http://www.abcam.com/protocols/dot-blot-
protocol
 http://feedforbiotech.blogspot.com/2011/03/dot-
blot.html
 https://www.thermofisher.com (far western)
 https://fr.wikipedia.org/wiki/Far-eastern_blot
 https://www.pathwayz.org/Tree/Plain/TRANS
GENESIS
 https://openi.nlm.nih.gov(mrna degradation)
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38.  http://www.tiem.utk.edu/~gross/bioed/web
modules/mRNA.htm
 http://bitesizebio.com/7206/introduction-to-
dna-microarrays/
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