Blotting techniques

1
Blotting Techniques
Asst. Prof. Om M. Bagade
M. Pharm, D. I. P. L
Department of
Pharmaceutics

Objective:
To make students understand regarding the Genetic
Engineering: Recombinant DNA technology with
respect to its methods for gene transfer, & different
Genetic Engineering techniques.
Outcome:
Students are familiar with the different DNA
transfer techniques & these method involves
separation, transfer and hybridization

What is blotting?
 Blots are techniques for transferring DNA ,
RNA and proteins onto a carrier so they can
be separated, and often follows the use of a
gel electrophoresis. The Southern blot is used
for transferring DNA, the Northern blot for
RNA and the western blot for PROTEIN.

TYPES OF BLOTTING
TECHNIQUES
Blotting
Techniques
Southern Blot
It is used to detect
DNA.
Northern Blot
It is used to detect
RNA.
Western blot
It is used to
detect protein.

SOUTHERN BLOTTING
 Professor Sir Edwin Southern,
Professor of Biochemistry and Fellow
of Trinity developed this method in
1975.
 Southern won the Lasker Award for
Clinical Medical Research prize for
the method of finding specific DNA
sequences he developed this procedure
at Edinburgh University more than 30
years ago. The technique is known as Professor Sir Edwin Southern

Cont….
 This method Involves separation, transfer and
hybridization.
 It is a method routinely used in molecular biology
for detection of a specific DNA sequence in DNA
samples. The DNA detected can be a single gene, or
it can be part of a larger piece of DNA such as a viral
genome.

Cont….
 Southern blotting combines agarose gel electrophoresis
for size separation of DNA with methods to transfer the
size separated DNA to a filter membrane for probe
hybridization.
 The key to this method is Hybridization.
 Hybridization - Process of forming a double-stranded
DNA molecule between a single-stranded DNA probe and
a single-stranded target patient DNA.

PRINCIPLE
1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the
molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected corresponds
to the location of the immobilized target molecule.

Whatman 3MM paper
Nitrocellulosemembrane

Steps in Southern Blotting
1. Digest the DNA with an
appropriate restriction
enzyme.
2.The complex mixture of
fragments is subjected to gel
electrophoresis to separate
the fragments according to
size.

Cont….
3.The restriction fragments
present in the gel are
denatured with alkali and
transferred onto
4. a nitrocellulose filter or
nylon membrane by
blotting.
 This procedure preserves
the distribution of the
fragments in the gel,

Cont….
5.The filter is incubated under
hybridization conditions with a
specific radiolabeled DNA
probe.
 The probe hybridizes to the
complementary DNA
restriction fragment.

Cont….
6. 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.

APPLICATIONS
 Southern blots are used in gene discovery ,
mapping, evolution and development studies,
diagnostics and forensics (It is used for DNA
fingerprinting, preparation of RFLP maps)
 Identification of the transferred genes in
transgenic individuals, etc.

 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.

Northern Blotting
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

Steps involved in Northern blotting
1. RNA is isolated from several
biological samples (e.g.
various tissues, various
developmental stages of
same tissue etc.)
* RNA is more susceptible
to degradation than DNA.

Cont……
2. Sample’s are loaded on
gel and the RNA samples
are separated according to
their size on an agarose
gel .
 The resulting gel following
after the electrophoresis

Cont……
3. The gel is then blotted
on a nylon membrane
or a nitrocellulose
filter paper by creating
the sandwich
arrangement.

Cont……
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

Cont……
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.

APPLICATIONS
 A standard for the study of gene expression at the
level of mRNA (messenger RNA transcripts)
 Detection of mRNA transcript size
 Study RNA degradation
 Study RNA splicing
 Study RNA half-life
 Often used to confirm and check transgenic /
knockout mice (animals)

Disadvantage of Nourthern plotting
1.The standard northern blot method is relatively less
sensitive than nuclease protection assays and RT-
PCR
2. Detection with multiple probes is a problem
3. If RNA samples are even slightly degraded by RNases,
the quality of the data and quantitation of expression
is quite negatively affected.

Western blotting
 Western blotting (1981) is an Immunoblotting
technique which rely on the specificity of binding
between a protein of interest and a probe (antibody
raised against that particular protein) to allow
detection of the protein of interest in a mixture of
many other similar molecules.
 The SDS PAGE technique is a prerequisite for

Steps in western blotting
1. A protein sample is
subjected to
electrophoresis on an SDS-
polyacrylamide gel.
2. Electroblotting transfers
the separated proteins from
the gel to the surface of a

Cont…
3. The blot is incubated with a generic protein
(such as milk proteins or BSA) which binds
to any remaining sticky places on the
nitrocellulose.
4. An antibody that is specific for the protein of
interest (the primary antibody - Ab1) is
added to the nitrocellulose sheet and reacts
with the antigen. Only the band containing
the protein of interest binds the antibody,

Cont…
5. After washing for removal of non-
specifically bound Ab1, second
antibody (Ab2)is added, which
specifically recognizes the Fc
domain of the primary antibody and
binds it. Ab2 is radioactively
labeled, or is covalently linked to a
reporter enzyme, which allows to
visualize the protein-Ab1-Ab2

Applications
1. The confirmatory HIV test
2. Western blot is also used as the definitive test for
Bovine spongiform encephalopathy (BSE(
3. Some forms of Lyme disease testing employ
Western blotting .

What Do you Think about following………..
Q.1.What do you mean by Blotting Techniques?
Q.2. Give detail explanation about Southern, Western
&
Northern Blotting Techniques?
Q.3. Give applications of each techniques?

Blotting techniques

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