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Immunoblotting techniques: Principle, methodology and application.

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Immunoblotting techniques: Principle,
methodology and application.

RAHUL DEV BAIRAGI

 BLOTTING
&
BLOTTING TECHNIQUES

 Visualization of specific DNA, RNA & protein among many

thousands of contaminating molecules requires the convergence of
number of techniques which are collectively termed BLOT
transfer.
 Blotting technique Western blot It is used to detect protein.
 Northern Blot It is used to detect RNA.
 Southern Blot It is used to detect DNA.
 IMMUNOBLOTTING

 A laboratory procedure, in which proteins that have been separated

by electrophoresis are transferred to a membrane of nitrocellulose
or another material and are identified by their reaction with labeled
antibodies.
 Immunoblotting allows detection of a protein antigen immobilized
on the protein-retaining membrane support such as nitrocellulose
or polyvinylidene fluoride (PVDF). The detection of the protein of
interest relies on the binding of an antibody that specifically
recognizes the protein of interest exposed on the membrane.
 IMMUNODETECTION

 The identification of specific antibodies is possible after the

separation and blotting of proteins. Specific antibodies (mono or
polyclonal) bind to "their" band of proteins. Unspecifically binding
antibodies are removed by washing with detergent-containing buffers.
Additionally, unspecific binding pockets can be blocked before the
addition of specific antibodies.

 Primary antibodies are usually applied first, which are then

recognized by a secondary antibody. The secondary antibody is
conjugated with color, radioactivity or an enzyme for detection.
Biotin-conjugated antibodies are also used for this purpose.
WESTERN BLOTTING (IMMUNOBLOT)
 The western blotting (immunoblot) method entails various
advantages as compared to other immunosorbent assays (ISAs), like
for example ELISA. Western blotting (immunoblot) expands on the
idea of ELISA by allowing separation of the protein mix by size,
charge, and/or conformation.
 The described method of stripping allows for the detection of
several targets, contrary to ELISA where only one protein can be
detected. As the gel electophoreis of proteins separates the proteins
into bands, one can determine the size of the target
protein/polypeptide.
 It is also possible to (semi-)quantify the protein of interest by
running an internal quantity standard in parallel with the samples in
the gel. Similarly, the protein content of the samples can be compared
("sample A contains more protein than sample B").
 PRINCIPLE OF WESTERN BLOTTING

 Western blotting technique is used for identification of particular

protein from the mixture of protein.
 In this method labelled antibody against particular protein is used
identify the desired protein, so it is a specific test. Western blotting
is also known as immunoblotting because it uses antibodies to
detect the protein.
 Western blot is the analytical technique used in molecular biology,
immunogenetics, and other molecular biology to detect specific
proteins in a sample of tissue homogenate or extract. Western
blotting is called so as the procedure is similar to Southern
blotting.
METHODOLOGY OF WESTERN BLOTTING

 There are six steps involved in western blot.

1. Sample preparation

2. Gel electrophoresis

3. Proteins transfer

4. Blocking

5. Antibody incubation and

6. Proteins detection & Visualization

 SAMPLE PREPARATION

 Proteins can be extracted from different samples, such as

tissues or cells. Since tissue samples display a higher degree of
structure, the tissues are first broken down by the mechanical
invention, such as homogenizer or sonication.
 Protease and phosphatase inhibitors are commonly used to
prevent the digestion of the sample at cold temperatures.
 After protein extraction, it is important to detect the
concentration of proteins, which permits the mass of proteins
loaded into each well. And a spectrophotometer is often used
for proteins concentration.
 GEL ELECTROPHORESIS
 The most commonly used gel is polyacrylamide gels (PAG) and
buffers loaded with sodium dodecyl sulfate (SDS). Western blot
uses two types of agarose gel: stacking gel that is used for
concentrate all proteins in one band and separating gel that allows
for separating proteins according to their molecular weight. Smaller
proteins migrate faster in SDS-PAGE when a voltage is applied.
 PAGE can separate proteins ranging from 5 to 2,000 kDa according
to the uniform pore size which is controlled by the Different
concentration of PAG. Typically separating gels are made in 5%,
8%, 10%, 12% or 15%.
 When we choose the appropriate percentage of the separating gel,
we should consider the size of the target proteins. The smaller the
known weight of proteins is the higher percentage of gels should be
used.
 PROTEINS TRANSFER
 After separating proteins by gel electrophoresis, proteins are moved
from within the gel onto a solid support membrane to make the
proteins accessible to antibody detection.
 The main method for transferring proteins is called electroblotting,
which uses an electric field oriented perpendicular to the surface of
the gel, to pull proteins out of the gel and move into the membrane.
 It can be done semi-dry or wet conditions, while wet conditions are
usually more reliable as it is less likely dry out the gel. As shown in
the left figure, the membrane is placed between the gel surface and
filter.
 The transfer sandwich is created as follows: a fiber pad (sponge),
filter papers, the gel, a membrane, filter papers, a fiber pad
(sponge).
 BLOCKING

 Blocking is an important step in the western blot to prevent

antibodies from binding to the membrane non-specifically.
 The most commonly used typical blockers are BSA and non-fat dry
milk. When the membrane is placed in the dilute solution of
proteins, the proteins attach to all places in the membrane where the
target proteins have not attached.
 In this way, the “noise” in the final product of the western blot can
be reduced and result in clearer results.
 ANTIBODY INCUBATION

 After blocking, the primary antibody binds to target protein when

the primary antibody is incubated with the membrane. The choice
of a primary antibody depends on the antigen to be detected.
 Washing the membrane with the antibody-buffer solution is helpful
for minimizing background and removes unbound antibodies. After
rinsing the membrane, the membrane is exposed to the specific
enzyme conjugated secondary antibody.
 When performing secondary antibody incubation, the labeled
secondary antibody can bind to the primary antibody which has
reacted with target proteins. Based on the species of the primary
antibody, we can choose the appropriate secondary antibody.
 PROTEIN DETECTION & VISUALIZATION

 A substrate reacts with the enzyme that is bound to the secondary

antibody to generate colored substance.
 It enables us to know the densitometry and location of the targets
protein. And the size approximations are taken by comparing the
proteins bands to the marker.
 There are several detection systems are available for protein
visualization, such as colorimetric detection, chemiluminescent
detection, radioactive detection, and fluorescent detection.
 The electrochemiluminescence (ECL) system is the most common
detection method.
 APPLICATION OF WESTERN BLOTTING
 To determine the size and amount of protein in given sample.
 Disease diagnosis: detects antibody against virus or bacteria in serum.
 Useful to detect defective proteins. For eg Prions disease.
 Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis
B and Herpes.
 Identification of a specific protein in a complex mixture of proteins.
In this method, known antigens of well-defined molecular weight are
separated by SDS-PAGE and blotted onto nitrocellulose.
 Estimation of the size of the protein as well as the amount of protein
present in the mixture.
 It is most widely used as a confirmatory test for the diagnosis of HIV,
where this procedure is used to determine whether the patient has
antibodies that react with one or more viral proteins or not.
 Demonstration of specific antibodies in the serum for diagnosis of
neurocysticercosis and tubercular meningitis.
 DISADVANTAGES
 A disadvantage of western blotting (immunoblot) is that it is

 Time-consuming (compared to ELISA).

 High demand in terms of experience of the experimenter.

 Additionally, it requires optimizing the experimental

conditions (eg. Protein isolation, buffers, type of
separation, gel concentration, etc.).
FLOW DIAGRAM
 CONCLUSION
The western blot (sometimes called the protein immunoblot), or
western blotting, is a widely used analytical technique in molecular
biology and immunogenetics to detect specific proteins in a sample of
tissue homogenate or extract. Western blot technique uses three
elements to achieve its task of separating a specific protein from a
complex: separation by size, transfer of protein to a solid support, and
marking target protein using a primary and secondary antibody to
visualize. Other related techniques include dot blot analysis,
quantitative dot blot, immunohistochemistry and immunocytochemistry,
where antibodies are used to detect proteins in tissues and cells by
immunostaining, and enzyme-linked immunosorbent assay (ELISA).

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