1. The document discusses various blotting techniques including Southern blotting, Northern blotting, and Western blotting. It provides a brief history and overview of each technique. 2. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting detects proteins. The techniques involve separating biomolecules by electrophoresis, transferring them to a membrane, then using a probe for detection. 3. The document outlines the basic methodology for each type of blotting. This includes separation, transfer to a membrane, blocking, hybridization with a probe, washing, and detection. Each technique allows for the analysis of biomolecules and has various applications in research.

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SL NO. TOPIC
1. HISTORY
2. DNA HYBRIDIZATION
3. INTRODUCTION
4. SOUTHERN BLOTTING
5. NORTHERN BOLTTING
6. WESTERN BLOTTING
-Far Western Blotting
7. EASTERN BLOTTING
8. FAR EASTERN BLOTTING
9. DOT BLOT
10. CONCLUSION
11. REFERENCES

2. HISTORY
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3. 1963-Nygaard and Hall: Shown that single stranded DNA can be
immobilized on nitrocellulose filters.
1966 &1965-Denhardt and Spiegelman: Nucleic acid thus fixed can
be detected with exquisite sensitivity by hybridization to radiolabelled
probes.
1970s: Possibility of mapping whole genomes arose along with the era
of rDNA and gene cloning. Thus need to find a single gene among
thousands of fragments of DNA was needed.
1975-Edward Southern: Powerful DNA transfer and probing
techniques(Southern Blotting).
1977-George Stark & colleagues: Detection of RNA (Northern
Blotting)
1979-Stark developed early protein blotting.
Harry Towbin gave faster and simpler approach.
W. Neal Burnette’s (1981) technique named Western Blotting.
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6. Formation of stable base pairs between probe and target DNA.
PREPARE TARGET DNA
PREPARE PROBE DNA
HYBRIDISATION
Probe
(labelled)
Target
Sequence Hybrid 6The Oxford College of Science

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8. Blotting is a method of putting DNA, RNA or Proteins onto a
membrane for further studies and detection.
Southern Blotting: DNA is detected with a hybridization DNA
or RNA probe.
Northern Blotting: RNA is detected with a hybridization DNA or
RNA probe.
Western Blotting: Protein is detected with a complementary
antibody.
The three blotting techniques have similar methodology.
Molecules separated by electrophoretic procedures are transferred
to membranes that is specially suited to support the detection of
fragments with a particular DNA sequence, single species of RNA
or proteins.
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10. The first type of blotting to be discovered.
Plays significant role in Recombinant DNA Technology as well as
Molecular Biology.
Used to detect target DNA in a sample.
Sample Target DNA
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11. FLOW CHART:-
Preparation of sample & running the
agarose gel.
Southern Transfer.
Probe preparation.
Prehybridization.
Hybridization.
Post Hybridization Washing.
Signal Detection.
Isotope
Non-isotope
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12. Schematic
Diagram.
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13. Gel Electrophoresis (Size Separation):
Size based separation in an electric field
Sample Preparation
Isolation of DNA by common extraction protocol
Purified DNA partially digested by restriction endonuclease
Linearized DNA loaded.
Aragose Electrophoresis
•Pretreatment of gel with
-Concentrated HCl
-Alkaline solution
• DNA is negatively charged thus moves from
cathode to anode.
•Sorter fragments move faster.
•Agarose 0.5% to 2%.
•Voltage of about 100mV
•Buffers used are TAE and Sodium Borate
•P32 labeled marker as ladder 13The Oxford College of Science

14. Step II
Southern Transfer:
Transfer DNA from gel to solid support.
SOLID SUPPORT
Nitrocellulose Membrane
-Nucleic acids more than 400 bases are inefficiently bound.
- Attachment by hydrophobic interactions.
-Become brittle while baking in vacuum.
- Care required for storing.
Nylon
-Buffers of low ionic strength can be used.
- Transfer can be carried out electrophoretically.
-Two types a)Neutral b)Positively charged (amines).
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15. NITROCELLULOSE
MEMBRANE
NYLON MEMBRANE
Hydrophobic binding. Covalent binding.
Fragile Durable
>200-300 bp probe
length
<200-300 bp probe can
be used
Lower background noise Higher.
Cannot be exposed to
basic solution
Can be exposed.
Not easily reprobed Can be easily reprobed
several times.
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16. Transfer of electrophoretically separated DNA from
gel to a 2D support is the key step.
1.Upward Capillary Action.
Rate of transfer depends on size of DNA and concentration of gel.
Gel dehydrates eventually.
DEPURINATION
-dilute HCl and then strong base.
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17. 2. Downward Capillary Action.
•Alkaline buffer (NaOH)
•Nylon membrane (generally charged)
•Rapid
•More efficient
3.Simultaneous Transfer To Two
Membranes.
•Target DNA fragments higher in
concentration.
•Transfer buffer is just the liquid
trapped in gel
•Efficiency of transfer poor.
•Used for plasmids,
bacteriophages, cosmids and
genome of simple organisms
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(Saccharomyces cerevisiae & Drosophilla)
•Not sensitive enough to analyze complex mammalian genome.

18. 4. Electrophoretic Transfer.
•Charged nylon membrane
•Analysis of small fragments of DNA(~50kb)
separated through polyacrylamide gels
•Gel neutralised
•1X TBE buffer used.
5.Vacuum Transfer.
•Rapid
•Gel is placed in contact with
membrane supported on a porous
screen over a vacuum chamber.
•Buffer drawn from an upper
reservoir, elutes nucleic acids from
the gel & deposits them on the
membrane.
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19. Step III
Blocking (Prehybridization)
After the southern transfer the membrane is generally
baked at 80ºC for 2 hours or UV (in nylon) for
permanent attachment.
The membrane has high affinity for proteins and
nucleic acids. Thus non specific binding between
probes and the material has to be prevented by
blocking.
This is done by soaking the membrane in a
solution containing high concentrations of DNA
(example herring or salmon sperm DNA) or
ficoll.
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20. Step IV Hybridization
Single stranded DNA to be detected forms hybrid with
complementary single stranded probe that can be detected
either by radioactivity (P32) or by chemiluminiscence
(digoxigenin).
Quantitative analysis:
-Darker band: complete
hybridization
-Lighter band: Incomplete
hybridization
Stringency is determined by the
hybridization temperature and salt
concentration in buffer.
Under gentle agitation, with a small
amount of detergent hybridization is
allowed for hours(in a closed bag at
about 68ºC). 20The Oxford College of Science

21. Step V
Step VI
Washing
Detection
Membrane is rinsed several times with different buffers to
remove any unbound nucleic acid probe, in order to avoid
unspecific background signals.
•If probe is radioactive, visualization is
done on x-ray film by autoradiography.
•Membrane is pressed against the film, which in turn is
exposed, for a few minutes to weeks.
•Non-radioactive methods are safer, quicker & cheaper.
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22. 1. Detection of an RFLP by Southern Blotting.
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23. 2.Detection of DNA defect & DNA segment
(Gene Defect)
Eg-Detection of the sickle cell globin gene
TYPE OF Hb AMINO ACID SEQUENCE
NUCLEOTIDE SEQUENCE
A -Pro-Glu-Glu-
-CCT-GAG-GAG-
S -Pro-Val-Glu-
-CCT-GTG-GAG-
Using restriction enzymes we can prove that they are different
types of genes.
•Normal DNA- 2 segments
•Sickle cell- 1
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25. 3. Used for homology based cloning on the basis of amino
acid sequence of protein product of the gene.
Oligonucleotides are designed similar to target sequence.
Chemically synthesized, radio labeled and used to screen a
DNA library. Sequences that hybridize with the hybridization
probe are further analyzed.
4. To identify methylated sites in a particular gene. Msp I and
Hpa II recognize and cleave the same sequence.
Msp I DNA unmethylated
Hpa II C has to methylated
5. To isolate specific DNA in a DNA sample (for RDT).
6. Used in phylogenetic analysis.
7. Diagnosis of infectious diseases.
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26. 8. In DNA fingerprinting
(Paternity and maternity testing,
Criminal identification and
Forensics and personal identification).
9. To follow inheritence of certain selected genes.
10. To know restriction sites for restriction endonucleases.
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27. 1) Laborious
2) Time consuming
3) More amount of DNA required
4) 3µl DNA per sample is costly.
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29. DETECTION OF RNA USING SS PROBE HYBRIDIZATION
TECHNIQUE
•Most of the procedure remains same as Southern Blotting
but the only problem that arises is that the single stranded
RNA tend to re-anneal themselves in a 2º & 3º structures.
•These structures are resolved using formaldehyde or
formalin.
•No alkali treatment required.
•Using this technique, we can come to know about the
presence of our transcripts & expression of gene in DNA,
along with looking for RNA splicing, maturation and
degradation.
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30. FLOW CHART:-
Preparation of RNA sample &
running the agarose gel.
Northern Transfer.
Probe preparation.
Prehybridization.
Hybridization.
Post Hybridization Washing.
Signal Detection.
Isotope
Non-isotope
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31. Sample Preparation: Pretreated with formaldehyde to
prevent the formation of base paired secondary
structure.
NOTE
• Care should be
taken as RNA is
less stable
• No solution should
contain RNAse
• DNAse should be
added
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32. PROBLEM
•Amount of sample to be loaded may vary as
there are different types of RNAs
•If mRNA is abundant, we can take the whole
RNA in count
Or else we take poly(A) content as mRNA
contains poly(A) tails.
•This should be followed or the band may vary.
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•Electroblot- reliable and
efficient.
•Hybridization 60ºC for less
than 2 hours.
•EDC cross linking (UV
cross linking)

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APPLICATIONS
1)To understand gene expression at the level of
mRNA.
2)mRNA transcript size
3)RNA degradation
4)RNA splicing
5)RNA half life
6)Used to confirm or check transgenic or knockout
mice.

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LIMITATIONS
1) Standard Northern Blotting is relatively ineffective than
nuclease protection assay or RT-PCR (Reverse
Transcription).
2) Detection with multiple probes become a problem
3) RNA is fragile and can be easily degraded by RNAse.
Thus the quality of data & quantification of expression
quite negatively affected.

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•Used for the detection of proteins.
•Also called Immunoblotting
•4-5 days to get complete result.
•Two types
-Direct: Single antibody
-Indirect: 1º and 2º antibody used (in practice) .
Steps:
Sample Preparation
SDS-PAGE
Blocking
Probing
Washing
Detection

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1. Sample preparation:
Cells lysed in extraction buffer containing proteinase
inhibitors
Samples cooled or frozen & homogenized using
mechanical force.
Centrifugation employed for protein purification.
Samples have boiled for one to five minutes in a denaturing
buffer (eg. Laemmli’s buffer)

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2. SDS-PAGE
Separation of proteins by electrophoresis.
3.Electrotransfer
Transfer of proteins to PVDF
(Polyvinyldienefluoride) membrane.
4.Blocking
• 5% nonfat dry milk or 3% BSA.
• Presence of detergent (Tween 20) at 0.05% is also very important.

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5. Probe
• Primary (rabbit anti human β actin) and
Secondary antibody (HRP-conjugated anti rabbit) employed
• Monoclonal antibody
• Primary antibody concentration 0.5-5µg/ml
6. Enzyme
Luminol is degraded
by secondary antibody
HRP to give
luminescence(425 nm)
7. Storing
Washed and exposed
to x-ray film so as to
study later

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APPLICATIONS
1) Verify the presence of a protein.
2) Find the relative amount of a protein in different
samples.
3) Analyze protein-protein interactions.

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1) Highly sensitive.
As little as 1.5ng of an average sized protein can be
detected.
2) Comparatively quick methodology.
3) Quantification can be done by densitometries and
integrating areas under the peak.

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LIMITATIONS
1) Many steps where errors may occur.
2) Significant amount of sample needed.
3) Accurate quantification is vey difficult.
4) Time consuming protocol.
5) Tertiary structure destroyed and therefore relevant
epitope recognized by primary antibody may not be
understood.

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•Detects protein-protein interactions in vitro.
• Proteins in a cell lysate containing prey proteins are firstly
separated by SDS -PAGE.
Transferred to a membrane (as in a standard WB)
Proteins on the membrane are then denatured and renatured.
Membrane is then blocked and probed, usually with
purified bait protein(s).
The bait proteins are detected as spots in the membrane
where a prey protein is located. (As bait proteins and the
prey protein form a complex)

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•Compared with other biochemical binding assays, Far WB
allows prey proteins to be endogenously expressed without
purification.
•Application includes:
1) receptor-ligand interactions study
2) screen libraries for interacting proteins and
3) identify protein-protein interactions without using antigen-
specific antibodies.
•Typically, 2-3 days are required to carry out the experiment.

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•Used to analyze protein post translational modifications
(PTM) such as lipids, phosphomoieties and glycoconjugates.
It is most often used to detect carbohydrate epitopes.
• Considered an extension of the of Western blotting.
•Multiple techniques have been described by the term Eastern
blotting, most use proteins blotted from SDS-PAGE gel on to
a PVDF or nitrocellulose membrane.
•Transferred proteins are analyzed for post-translational
modifications using probes that may detect lipids,
carbohydrate, phosphorylation or any other protein
modification.

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APPLICATIONS
 Detection of protein modifications in two bacterial
species Ehrlichia- E. muris and IOE.
Cholera toxin B subunit (which binds
to gangliosides), Concanavalin A(which detects mannose-
containing glycans) and nitrophospho molybdate-methyl
green (which detects phosphoproteins) were used to detect
protein modifications.
The technique showed that the antigenic proteins of the
non-virulent E.muris is more post-translationally modified
than the highly virulent IOE

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Post-translational modifications occurring at the N-
terminus of the amino acid chain play an important role in
translocation across biological membranes.
These include secretory proteins in prokaryotes and
eukaryotes and also proteins that are intended to be
incorporated in various cellular and organelle membranes
such as lysosomes, chloroplast, mitochondria and plasma
membrane.
Expression of post translated proteins is important in several
diseases.

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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 (HPTLC).
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 in minutes.
Name in dual reference to Southern
blot & Japan.

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•Purification of glycosphingolipids and 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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•Simplification of blotting techniques.
• In a dot blot the biomolecules to be detected are not first
separated by electrophoresis. Instead, a mixture containing
the molecule to be detected is applied directly on a membrane
as a dot, and then is spotted through circular templates directly
onto the membrane or paper substrate.
• It offers no information on the size of the target biomolecule.
Furthermore, if two molecules of different sizes are detected,
they will still appear as a single dot.
•Can only confirm the presence or absence of a biomolecule.

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• Blotting is a powerful and sensitive technique for identifying
the presence of specific biomolecules within a sample.
•The first of these techniques developed was the Southern blot
to detect specific DNA sequences by Dr. Edwin Southern,
1975.
•Subsequently, the method was modified to detect other
targets.
>Northern blot (for detection of RNA)
>Western blot (for detection of protein)
>Far western blot (Protein-protein interaction)
>Eastern blot (for detection of post translationally
modified proteins)
>Far eastern blot (lipid analysis)
>Southwestern blot (for detection of DNA binding
proteins)

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• Blotting techniques have been widely employed for more than 30
years and have provided the foundation of our understanding of molecular
biology.
• However, these techniques have been largely—and in some cases
completely—usurped by new technologies .
• Southern blots have been replaced by multiple techniques.
1. Real-time PCR boasts incredible sensitivity; theoretically, this
method is able to detect even a single copy of the target sequence
and compare relative copy numbers across samples rapidly and
reliably, with little technical expertise required.
2. Fluorescent in situ hybridization (FISH) allows detection of
specific sequences within a tissue sample with high sensitivity and
precise localization.
• Northern blots have given way to reverse-transcription PCR, again a
more sensitive and more user-friendly technique

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BOOKS
1. Molecular cloning, A laboratory Manual; Volume I;
Third Edition; Sambrook and Russel; Cold Spring
Harbour Laboratory Press, New York; Pages 6.33-6.58
and 7.42-7.46
WEBSITES
1. file:///F:/blotting/for%20conclusion.htm
2. http://en.wikipedia.org/wiki/Dot_blot
3. http://en.wikipedia.org/wiki/Far-Eastern_blotting
4. http://en.wikipedia.org/wiki/Eastern_blot#cite_note-thomas-14
5. http://www.ncbi.nlm.nih.gov/pubmed/18079728
6. http://books.google.co.in/books?id=ZDgh_TaLzNEC&pg=PA48&lpg=PA48&dq
=blotting+techniques+history&source=bl&ots=BUsrvNI6_K&sig=I1NbWeJ5R
Ux6SQ3LTo3k4aL-
Kwg&hl=en&sa=X&ei=E4kAVL6zKIehugT39YL4BQ&ved=0CF4Q6AEwBw#
v=onepage&q=blotting%20techniques%20history&f=false
7. http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction#
Application

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