SOUTHERN AND

WESTERN BLOTTING
HISTOR
Y
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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. The Oxford College of Science 3
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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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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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 FLOW CHART:-

Preparation of sample & running the

agarose gel.

Southern Transfer.

Probe preparation. Isotope

Non-isotope
Prehybridization.

Hybridization.

Post Hybridization Washing.

Signal Detection.
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Schematic
Diagram.
12
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 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
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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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NITROCELLULOSE NYLON MEMBRANE
MEMBRANE
Hydrophobic binding. Covalent binding.

Fragile Durable

>200-300 bp probe <200-300 bp probe can

length be used
Lower background noise Higher.

Cannot be exposed to Can be exposed.

basic solution
Not easily reprobed Can be easily reprobed
several times.

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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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 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
(Saccharomyces cerevisiae
& Drosophilla) The Oxford College of Science 15
 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
membrane.
the gel & deposits them on the
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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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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 detergents
hybridization i allowed for
hours(in a closed bag at about The Oxford College of Science 20
Step Washing

V
Membrane is rinsed several times with different buffers to
remove any unbound nucleic acid probe, in order to
avoid unspecific background signals.

Step Detection

•VI
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.
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1) Laborious

2) Time consuming

3) More amount of DNA

required

4) 3µl DNA per sample is costly.

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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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 APPLICATION
S
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

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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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•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) The Oxford College of Science 55
•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=P
A48&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
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