Western blotting test

Brief introduction of Western blotting ,Principle and Procedure..

1. Western blotting Presented by : Rajesh B.K. BScMLT 4 th Batch Chitwan Medical College 4/26/2017 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. 4/26/2017 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 . 4/26/2017 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. 4/26/2017 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. 4/26/2017 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. 4/26/2017 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. 4/26/2017 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. 4/26/2017 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 4/26/2017 9

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