Molecular biology

1. WESTERN
BLOTTING
B Y
MUSKAN
21018
M.Sc. BIOTECHNOLOGY 1

2. BLOTTING
◦ Blots are techniques for transferring DNA , RNA and Proteins onto aa carrier so
they can be separated , and often follows the use of gel electrophoresis. The
Southern blot is used for transferring DNA, the Northern blot for RNA and the
Western blot for PROTEINS.

3. BLOTTING
TECHNIQUE
SOUTHERN BLOTTING NORTHERN BLOTTING
WESTERN
BLOTTING
TYPES OF BLOTTING TECHNIQUES

4. WESTERN BLOTTING
◦ Western blotting is widely used analytical technique in molecular biology to
detect specific protein in a sample of tissue homogenate or extract.
◦ It works on the principle of gel electrophoresis.
◦ Proteins are separated based on their size on polyacrylamide gel

5. PRINCIPLE
◦ Western blotting is an Immunoblotting technique which rely on the principle
of binding between a molecule of interest and a probe to allow 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 a probe is
typically an antibody raised against that particular protein.
◦ The SDS PAGE technique is a prerequisite for Western blotting.

6. PROCEDURE
◦Tissue preparation
◦Gel electrophoresis
◦Transfer
◦Blocking
◦Detection
◦Analysis

7. TISSUE PREPARATIONS
◦ Samples are taken from whole tissue or from cell culture.
◦ It should be noted that bacteria, virus or environmental samples can be the
source of protein and thus western blotting is not restricted to cellular studies
only.
◦ Assorted detergents, salts, and buffers may be employed to encourage lysis of
cells and to solubilize proteins.
◦ Tissue prepration is often done at cold temperatures to avoid protein
denaturing.

8. Sample preparation
Ultra sonication for cell
suspension
Mechanical
homogenization for Plant
animal tissue
Enzymatic Digestion for
Bacterial , yeast, and
fungal cells
Detergent lysis for tissue culture
SAMPLE PREPARATION

9. GEL ELECTROPHORESIS
◦ The proteins of the sample are separated using gel electrophoresis. Separation
of proteins may be by isoelectric point, molecular weight, electric charge or a
combination of these factors.
◦ The principle involved is the difference in the ELECTROPHORETIC MOBILITIES of
different proteins.

10. SDS PAGE SAMPLE PROCESSING

11. SAMPLE LOADING

12. TRANSFERRING
◦ In order to make the proteins accessible to antibody
detection , they are moved from within the gel onto a
membrane made of nitrocellulose or polyvinylidene
difluoride(PVDF)

13. TRANSFERRING
◦ The membrane is placed on the top of the gel, and a stack of filter paper
placed on the top of that. The entire stack is placed in a buffer solution which
moves up the paper by capillary action , bringing the proteins with it.
◦ Another method of transferring the protein is called electro blotting and uses
an electric current to pull proteins from the gel into the PVDF or nitrocellulose
membrane.

14. BLOCKING
◦ The membrane has the ability to bind to proteins . In this case both the target and the
antibodies are proteins and so there could be some unwanted binding.
◦ Blocking of non-specific binding is achieved by placing the membrane in a dilute
solution of protein- typically Bovine serum with minute percentage of detergent such
as Tween 20.
◦ The protein in the diluted solution attaches to the membrane in all the places where
the target proteins have not attached. Thus, when the antibody is added, there is no
room on the membrane for it to attach other than on the binding sites of the specific
target protein.

15. BLOCKING AGENTS
The most common blocking agents are :-
a) Bovine serum albumin (BSA)
b) Non fat milk
c) Casein
d) Gelatin
e) Dilute solution of Tween 20

16. DETECTION
There are two steps for detection of proteins-
A) PRIMARY ANTIBODY
◦ After blocking , a dilute solution of primary antibody is incubated with the
membrane under gentle agitation.
◦ The solution is composed of buffered saline solution with a small percentage
of detergent , and sometimes with powdered milk

17. DETECTION
◦ The antibody solution and the membrane can be sealed and incubated together for
anywhere from 30 minutes to overnight.
B) SECONDARY ANTIBODY
◦ After rinsing the membrane to remove unbound primary antibody, the membrane is
exposed to another antibody, directed at a species-specific portion of the primary
antibody.
◦ Several secondary antibodies will bind to one primary antibody and enhance the
signal.

18. DETECTION

19. ANALYSIS & IMAGING
◦ This is the last and major step of the western blotting technique.
◦ Detection of signals using either X –ray film, scanners or a CCD, results in one
or more visible proteins bands on the membrane image.
◦ The molecular weight of the protein can be estimated by comparison with
marker proteins and the amount of protein can be determined as this is related
to band intensity.
◦ Qualitative and quantitative analysis can be done in order to verify the
absence or presence of specific proteins of interest.

21. APPLICATIONS OF WESTERN
BLOTTING
◦ Analysis of IgG fractions purified from human plasma.
◦ Diagnosis of HIV by ELISA , involves the western blotting technique.
◦ Western blotting technique is also used to detect some forms of Lyme disease.
◦ Western blotting technique is used in defence tests for BSE, which is commonly called
as Mad Cow disease.
◦ Confirmatory test for Hepatitis-B involves western blotting.
◦ This technique is also employed in the Gene Expression Studies.

22. ADVANTAGES
◦ While ELISA being a non-specific test, Western blotting is a more specific test
for detection of HIV .
◦ It can detect one protein in a mixture of proteins while giving information
about the size of the protein and so is more specific.
◦ It also tells how much protein has accumulated in cells.

23. DISADVANTAGES
◦ Very delicate and time consuming process.
◦ If a protein is degraded quickly, western blotting wont detect it well.
◦ It might be costly.
◦ Well trained technicians are required for this technique.

24. REFERENCES
◦ https://www.slideshare.net/AkanshGoel5/western-blotting-technique
◦ https://www.slideshare.net/doctorrao/western-blotting
◦ https://youtu.be/Yh69yHJMWPc
◦ https://www.slideshare.net/vivekaiden/western-blotting-119614678

25. THANK YOU !