Introduction and Description to Western Blotting, Steps involved in Western Blotting- Sample Preparation, Protein Gel Electrophoresis, SDS-PAGE, Protein Transfer, Electrophoretic Protein Transfer, Transfer Sandwich Diagram, Blocking, Antibody Probing and Detection, Applications of Western Blotting.

1. TYBSc Microbiology,
Genetics & Molecular Biology-I (MB-332)

2. Blots are techniques for transferring DNA, RNA & proteins onto a
carrier so they can be separated, & often follows the use of gel
electrophoresis.

3. Western blotting is an important technique which is used in the field
of cell and molecular biology.
Western Blotting is used to identify specific proteins from a
complex mixture of proteins extracted from cells.
This technique uses three basic factors to separate proteins, that is:
1) separation on the basis of molecular size,
2) transfer to a solid support, and
3) marking target protein using a proper primary and secondary
antibody for proper visualization.
Western Blotting is also known as Immunoblotting technique.

4. Steps involved in Western Blotting are:
1.Sample Preparation
2.Protein Gel Electrophoresis
3.Protein Transfer
4.Blocking
5.Antibody Probing

5. It should be noted that cell cultures of bacteria, virus, whole tissues
or other environmental samples can be the source of protein.
Cell lysis agents including salts{NaCl, KCl, (NH4)2SO4}, buffers
(Tris–HCl, Sodium dihydrogen phosphate, Disodium hydrogen
phosphate) and detergents (SDS, ethyl trimethyl ammonium
bromide, Triton X-100) are used.
 After cell lysis, the protein extract is purified & further used in gel
electrophoresis.

6. The protein samples are separated into their component proteins by
gel electrophoresis.
Separation of proteins can be done on the basis of isoelectric point,
molecular weight, electric charge or a combination of these factors.
The principle is based on the difference in electrophoretic
mobilities of different proteins.
The most commonly used electrophoresis is SDS-PAGE – Sodium
dodecyl sulphate polyacrylamide gel electrophoresis.

7. SDS-PAGE separates proteins based on their molecular weight & all
the separated proteins contain a negative charge due to presence of
anionic detergent SDS.

9. Once proteins have been separated by SDS-PAGE, next step is to
transfer these proteins to a suitable membrane.
The membranes used for the transfer have high protein affinity,
excellent protein binding & retention capabilities.
Most commonly used membranes in Protein Transfer are-
Nitrocellulose, Polyvinylidene difluoride (PVDF).
Protein transfer is done for further detection & analysis, since the
membranes are thinner than gels, the epitopes of proteins are easily
accessible to bind to antibodies.

10. In this method, electric current is used to elute proteins from gel &
transfer them on the membrane.

11. Transfer Buffer- facilitates
movement of proteins from the gel
onto the protein.
Methanol which is present in
transfer buffer facilitates binding of
proteins on the membrane by
removing SDS from proteins.
Filter Paper- maintains uniform flow
of transfer buffer.
Sponge- maintains proper pressure
during the transfer.
At the end of this step, all the
proteins move to the membrane &
attaches tightly onto the membrane.

13. The process of blocking is done after protein transfer on the
membrane to avoid binding of antibodies onto the regions of
membranes where protein is absent.
Therefore, blocking agents are added to avoid the non-specific
binding of antibodies onto the membrane.
The most commonly used blocking agents are Bovine Serum
Albumin(BSA) & non-fat milk.
These agents specifically binds to the membrane without creating
disturbance on other proteins.

14. After addition of blocking agents, the membrane is incubated with
primary antibody which is specific to the target proteins.
Further, the membrane is incubated with labeled secondary
antibody, these antibodies bind to the already bound primary
antibodies.
The most common enzyme label used is Horseradish Peroxidase,
which acts on chemiluminescent substrates such as 4-chlor-1-
napthol (4CN).
The enzyme catalyzes oxidation of substrate forming an insoluble
visible purple product which helps in easy detection.

17. Western Blotting is a more specific test for HIV detection, it is applied in
a confirmatory HIV-test to detect anti-HIV antibody in a human serum
sample.
It is also used as definitive test for BSE- Bovine spongiform
encephalopathy, commonly called as mad cow disease.
Western immunoblotting technique is quantitative. Picogram quantities
of viral antigens and antibodies can be detected by this assay.
In veterinary medical field, western blot is sometimes applicated in the
medical diagnosis for FIV in cats.
Western blot is used as a confirmatory test for Hepatitis B infection.
Western blotting is also applied in combination with another technique of
ELISA in some forms of Lyme disease diagnosis test.

18. FIN
Presented By- Bishwarup SarkarPresented By- Bishwarup Sarkar