Brief introduction of Western blotting ,Principle and Procedure..

1. WESTERN BLOTTING
Presented by : Rajesh B.K.
BScMLT 4th Batch
Chitwan Medical College
5/11/2017
1

2. INTRODUCTION
 Named after scientist: Western Neal Burnette.
 Western blotting is a technique used to detect the
presence of a specific protein in a complex protein
mixture.
5/11/2017
2

3. PRINCIPLE OF WESTERN BLOTTING
 Western blotting is an Immunoblotting technique
which rely on the specificity 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 the probe is typically an antibody raised
against that particular protein.
 The SDS PAGE technique is a prerequisite for
Western blotting .
5/11/2017
3

4. 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 nitrocellulose
membrane.
5/11/2017
4

5. 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 , forming a layer of antibody
molecules.
5/11/2017
5

6. 5. After washing for
removal of non-specifically
bound Ab1, secondary
antibody (Ab2) is added.
•Ab2 specifically recognizes the
primary antibody and binds.
•Ab2 is radioactively labeled, or is
covalently linked to a reporter enzyme,
which allows to visualize the protein-Ab1-
Ab2 complex.
5/11/2017
6

7. Column 1: positive control; column 2: negative control; column 3-4: positive anti-HIV-1 serum;
column 5: positive anti-HIV-1 serum group O; column 6: positive anti-HIV-2 serum; columns 7-
10:sequential results from serum testing of a patient newly infected with HIV-1.
5/11/2017
7

8. APPLICATIONS
 The confirmatory HIV test .
 Used as the definitive test for Bovine spongiform
encephalopathy ( Mad cow disease).
 Some forms of Lyme disease testing employ
Western blotting.
 Western blot can also be used as a confirmatory test for
Hepatitis B infection.
 In veterinary medicine, western blot is sometimes used to
confirm FIV+ status in cats.
5/11/2017
8

9. REFERENCES:
 https://en.wikipedia.org/wiki/Western_blot
 http://www.antimicrobe.org/h28tab2.jpg
 https://www.youtube.com/watch?v=GUnOVfhb3K4
 Textbook of Microbiology and Immunology,
Subhash Chandra Parija
5/11/2017
9

10. 5/11/2017
10