• Blotting is the technique for transferring DNA, RNA

and proteins onto a carrier for the purpose of their

separation.
• It often follows the use of a gel electrophoresis.
 Blotting
technique

Southern Blot Northern Blot Western blot

It is used to It is used to
It is used to
detect RNA. detect protein.
detect DNA.
• Southern blot named after
Sir Edwin Southern
Developed in 1975 .

Professor Sir Edwin Southern

• The Southern blot is used to detect the presence of a
particular piece of DNA in a sample.

• The detected DNA can be a single gene, or it can be part of a

larger piece of DNA such as a viral genome.

• This method involves separation, transfer and hybridization.

• The key of southern blotting is Hybridization.

• Hybridization - Process of forming a double-stranded DNA

molecule from a single-stranded DNA probe and a single-
stranded target patient DNA.
1. The DNA (total DNA of an
organism) to be detected
or analysed is completely
digested with a restriction
enzyme .

2. The complex mixture of

DNA fragments is subjected
to gel electrophoresis to
separate the fragments
according to their size.
3. The restriction fragments
present in the gel are
denatured with alkali and
transferred onto a
nitrocellulose filter or
nylon membrane by
blotting.
This procedure preserves
the distribution of the
fragments in the gel,
creating a replica of the
gel on the filter.
4. The filter is incubated under
hybridization conditions with a
specific radiolabeled DNA
probe.

• The probe hybridizes to the

complementary DNA
restriction fragment.
5. Excess probe is washed
away and the probe bound
to the filter is detected by
autoradiography, which
reveals the DNA fragment
to which the probe
hybridized.
• Southern blots are used in several main areas including gene
discovery and mapping, evolution and development studies,
diagnostics and forensics.
• Southern blots allow investigators to determine the molecular
weight of a restriction fragment and to measure relative
amounts in different samples.
• Southern blot is used to detect the presence of a particular bit of
DNA in a sample .

• Analyze the genetic patterns which appear in a person's DNA.

• Used in DNA fingerprinting, genetic engineering, & forensic
science for tests such as: Paternity testing, Personal identification, Sex
determination.
• In regards to genetically modified organisms, Southern
blotting is used as a definitive test to ensure that a
particular section of DNA of known genetic sequence has
been successfully incorporated into the genome of the host
organism.

• Diagnosis of congenital inherited diseases such as

congenital adrenal hyperplasia (CAH).
• Identify mutations, deletions, and gene rearrangements.
• Used in prognosis of cancer and in prenatal diagnosis of
genetic diseases
• Leukemias
• Diagnosis of HIV-1 and infectious disease
• Northern blotting is a technique for detection
of specific RNA sequences.
• Northern blotting was developed in 1977 by
James Alwine, David Kemp and George Stark
at Stanford University and was named such by
analogy to Southern blotting .
1. Isolation of RNA: RNA is isolated from several biological
samples (e.g. various tissues, developmental stages of same
tissue etc.)
2. Electrophoresis: Sample’s are loaded on Agarose Gel
and the RNA samples are separated according to their
size on an agarose gel .
3. Blotting: The gel is then blotted on a nylon membrane
or a nitrocellulose filter paper by creating the sandwich
arrangement.
4. The membrane is placed
in a dish containing
hybridization buffer
with a labeled probe.
• Thus, it will hybridize to
the RNA on the blot that
corresponds to the
sequence of interest.

5. The membrane is
washed to remove
unbound probe.
6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is used).
In both cases this results in the
formation of a dark band on an X-ray
film.

• Now the expression patterns of the

sequence of interest in the different
samples can be compared.
• A standard for the direct study of gene expression at
the level of mRNA (messenger RNA transcripts).
• Detection of mRNA transcript size
• To Study RNA degradation
• To Study RNA splicing - can detect alternatively spliced
transcripts
• To Study RNA half-life
• It is a widely accepted straight-forward method

• It is used as a gold-standard for confirmation of particular RNA.

• It is a versatile protocol as it can allow the usage of many types of

probes (vs Real time PCR) including: radiolabeled and non-
radiolabeled, in vitro transcribed RNA and even oligonucleotides
such as primers.
• Use of radioactivity prevents its ease of performance, use
and disposal. New methods of non-radioactive detection
have been generated allowing non-radioactive detection.

• The whole process of northern blotting takes a long

time usually, from sample preparation to detection.

• If RNA samples are even slightly degraded by RNases, the

quality of the data and quantification of expression is
negatively affected.
• The standard northern blot method is relatively less
sensitive than nuclease protection assays and RT-PCR.
The sensitivity of northern blots may be increased with
the use of positively-charged nylon membranes, and use of
a highly specific antisense probe.

• Detection with multiple probes is a problem. Often, the

membranes must be stripped before hybridization and
detection with a second probe. This is a problem as harsh
conditions are required to strip off probes from the blot
and is also time consuming. Also, there is a limit to the
amount of times a blot may be stripped.
Western Blotting
or
Immunoblotting
• Western blotting is an Immunoblotting technique Introduced
by Towbin, et al. in 1979
• It is based on the specificity of binding between a molecule of
interest (protein) and a probe (antibody).
• This allows the detection of the molecule of interest in a
mixture of many other similar molecules.
• In Western blotting, the molecule of interest is a protein
and the probe is typically an antibody raised against that
particular protein.

• The SDS PAGE technique is a prerequisite for Western blotting.

1. Separation: A protein
sample is subjected to
electrophoresis on an SDS-
polyacrylamide gel.

2. Electro blotting:
Transfer the separated
proteins from the gel to
the surface of a
nitrocellulose
membrane by Electro-
blotting.
3. Blocking: Further, the blot (nitrocellulose paper) is incubated
with a generic protein (such as milk proteins or BSA). These
protein molecules binds to the remaining sticky places on the
nitrocellulose.
4. Primary antibody (Ab1) addition: An antibody that is specific
for the protein of interest is added to the nitrocellulose sheet so
that it will reacts with the antigen.
In this process, only the band containing the protein of interest
binds to the antibody and forms a layer of antibody molecules on
nitrocellulose membrane.

5. Washing: After that it is rinsed for several times for removal of

non-specifically bound primary antibody (Ab1).
6. Addition of radiolabelled
Secondary antibody (Ab2):
• The Ab1-antigen complex on the
nitrocellulose sheet is incubated
with a second antibody (Ab2), which
specifically recognizes the Fc
domain of the primary antibody and
binds to it.
• This Ab2 is radioactively labelled, or
is covalently linked to a reporter
enzyme, which allows to visualize
the protein-Ab1-Ab2 complex.
• Western blotting is a more specific test having ability to detect even
single protein from the mixture of various proteins.
• It also gives information about the size of the protein. This provides
more specific results than the ELISA protocol by providing additional
information regarding protein size and multiple bands.
• Western blot test is referred to as the 'Gold Standard' in protein
detection.
• Western blotting also gives idea about how much protein has
accumulated in cells.
• It is time-consuming (compared to ELISA)
• If a protein is degraded quickly, Western blotting won't
detect it well .

• Costly.

• It requires optimization of the experimental conditions

(i.e. protein-isolation, buffers, type of separation, gel-
concentration, etc.).
1. Western blot is used as a confirmatory test to detect HIV
antibodies in a human serum sample:
• Proteins from known HIV-infected cells are separated and blotted
on a membrane.
• then, the serum to be tested is applied in the primary antibody
incubation step;
• Free antibody is washed away, and a secondary anti-human
antibody linked to an enzyme signal is added.
• The stained bands indicates that the proteins to which the
patient's serum anti-bodies reacted.
• This confirms the HIV test
2. A Western blot is also used as the definitive test for
Bovine spongiform encephalopathy (BSE, commonly
referred to as 'mad cow disease).

3. It is also used for detection of Lyme disease.

 Southern blotting Western blotting

Molecule detected DNA (ds) Protein

Gel electrophoresis Agarose gel Polyacrylamide gel

Blotting method Capillary transfer Electric transfer

DNA Radioactive or non-

Probes radioactive primary antibody

Autoradiography
Chemiluminescent Chemiluminescent
Detection system Colorimetric Colorimetric