2. Outlines
■ What is Blotting ?
■ Introduction
■ Types of blotting
■ Why to do western blotting?
■ Principle
■ Reagents and materials required
■ Steps involved
■ Applications
■ Limitations
■ Conclusion
3. What is Blotting ?
■ Blotting is a technique of transferring proteins , DNA or RNA onto a Carrier
i.e. a nitrocellulose or PVDF (polyvinylidene fluoride ) or nylon membrane .
■ It refers to the transfer of biological samples from a gel to a membrane and
their subsequent detection on the surface of the membrane .
4. Introduction
■ This technique allow to identify a particular protein of interest among
many proteins in a sample .
■ Western Blotting is a widely used analytical technique used in
molecular biology , immunogenetics and other molecular biology
disciplines to detect specific proteins in a sample of tissue homogenate
or extract .
■ Western Blotting , sometimes called the protein blotting or
immunoblotting because an antibody is used to specifically detect its
antigen.
■ Western blotting was performed by the rapid method of Towbin et
al.(1979) to detect the antigens blotted on a Nitrocellulose membrane
with the use of an antibody.
5. Types of blotting techniques
BLOTTING
TECHNIQUES
SOUTHERN
BLOTTING
Uses to detect
DNA .
NOTHERN
BLOTTING
Used to detect
RNA .
WESTERN
BLOTTING
Used to detect
PROTEINS .
6. Why to do western blotting ?
■ To see if specific protein of interest is present in a sample .
■ To compare the amounts of a protein of interest among different
samples .
■ To see if the state of a protein changes under different biological
condition (example . In disease , stress etc.)
7. Principle
Western blotting ( protein blotting or immunoblotting ) is a rapid and sensitivity assay for detection and
characterization of proteins . It is based on the principle of immunochromatography where proteins are
separated into polyacrylamide gel according to their molecular weight .
The protein thus separated are then transferred for electro transferred onto nitrocellulose membrane and
are detected using specific primary antibody and secondary enzyme labeled antibody and substrate .
8. Reagents and Requirements
■ Nitrocellulose membrane
■ Plastic staining box
■ Electroblotting apparatus
■ Transfer buffer (500ml, pH7.6)
■ 10 X Tris buffered saline (TBS) (100ml , pH7.6)
■ Blocking solution
■ Washing buffer
■ Preparation of primary antibodies .
■ Preparation of secondary antibodies .
■ Color indicator solution.
10. Sample preparation
Samples may be taken from whole tissue or from cell culture . In most cases , solid tissues are
first broken down mechanically using a blender.
It should be noted that bacteria , virus or environmental samples can be the source of protein .
Assorted detergents , salts and buffers may be employed to encourage lysis of cells and to
solubilize proteins.
Sample preparation is often done at cold temperatures to avoid protein denaturation.
Sample preparation
Detergent lysis
For tissue
culture
Ultra
sonification
For
Cell suspension
Mechanical
Homogenizatio
n
For
Plant & Animal
tissues.
Enzymatic
digestion
for
Bacterial, yeast
and Fungal cells.
11. Step 1 : SEPARATE
■ After the sample have been prepared, the mixture is separated by using molecular
weight , using protein gel electrophoresis , which is an standard laboratory technique
by which charged protein molecules are transported through a matrix by an electric
field.
12. Step 2 : TRANSFER
■ Once the proteins have been separated in the gel, they must be transferred to a solid support
membrane (Nitrocellulose membrane or polyvinylidene difluoride (PVDF).
■ The process of transferring proteins from a gel to a membrane while maintaining their relative
positions and resolutions is known as blotting .
■ Using a traditional apparatus the protein transfer processes can take 30 to 60 minutes to complete .
However the new technologies have been developed that can cut the transfer time down to 7
minutes.
13. Step 3 : DETECTION
■ The membrane is ready for the detection and this process includes multiple steps such as :-
BLOCKING
PRIMARY
ANTIBODY
INCUBATION
WASHING
SECONDARY
ANTIBODY
INCUBATION
WASHING
INCUBATION
WITH
SUBSTRATES
CHEMILUMINESCENCE
DETECTION
IMAGE CAPTURE
AND
DOCUMENTATIO
N
15. APPLICATIONS
It can also identify a specific antibody in a mixture. In this case, known antigens of well-
defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. The
separated bands of known antigens are then probed with the sample suspected of containing
antibody specific for one or more of these antigens.
• It helps in the determination of size and amount of protein in the given sample.
• The most widely used application of Western blotting is in the confirmatory testing for HIV. It
determines whether the patient has antibodies against any viral proteins.
• It is useful in the detection of defective proteins in our body. E. g. Prions disease.
• It is applied as the definitive test for different diseases like Hepatitis B, Herpes, Lyme disease
etc.
16. LIMITATIONS
■ Very delicate and time consuming process ( A minute
imbalance at any level of the procedure can change the
results of the entire process)
Incorrect labeling of the protein (this can happen due to
the reaction of secondary antibody )
Causes errors in bands or no bands , due to insufficient
transfer.
Primary antibody is crucial .
17. CONCLUSION
Western blotting technique is simply a way to identify unknown
proteins on a polyacrylamide gel.
It is also called as protein blotting or immunoblotting .
It is widely used analytical technique in the fields of molecular
biology , immunogenetics and other biochemistry disciplines.
This technique is mainly used in the medical diagnostics i.e, in the
analysis of various kinds of diseases .
18. Reference
■ Kindt T. J, Goldsby R. A & Osbotne B. A, (sixth edition). Kuby
Immunology, New York , NY: W.H. Freeman and Company, 2007
■ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/#:~:text=Western%20bl
%20used,a%20band%20for%20each%20protein.
■ Laemmli, U. K. Cleavage of Structural Proteins During the Assembly of the Head
Bacteriophage T4. Nature 227, 680–685 (1970).
■ Köhler, G. & Milstein, C. Continuous Cultures of Fused Cells Secreting Antibody
Specificity. Nature 256, 495–497 (1975).
■ Southern, E. M. Detection of Specific Sequences Among DNA Fragments
Electrophoresis. J. Mol. Biol. 98, 503–517 (1975)
