Western Blotting ppt: Principle, Steps And Applications
What is Western Blotting:
In cell and molecular biology, Western blotting also referred to as immunoblotting or protein blotting, is a standard procedure. It is used to detect the presence of a particular protein in a complex mixture recovered from cells or tissue in its most basic form.
Principle of Western Blotting:
Western blotting (protein blotting or immunoblotting) is a fast and sensitive assay for detecting and characterizing proteins. It works on the basis of immunochromatography, which separates proteins into polyacrylamide gels based on their molecular weight.
The separated proteins are subsequently transferred or electro transferred onto nitrocellulose membranes, where they are identified using specific primary antibodies and secondary enzyme labelled antibodies and substrates.
Steps of Western Blotting:
Western blot completes into eight major steps:,
- sample preparation for protein extraction
- SDS -PAGE gel electrophoresis,
- Membrane transfer i.e Blotting can be done in two ways: electrical or capillary.
- BSA (blocking)
- The use of a primary antibody
- The introduction of a secondary antibody ( enzyme labelled anti Ab)
- Treatment with a particular substrate; for example, if the enzyme is alkaline phosphatase, the substrate is p-nitro phenyl phosphate, which produces colour.
- Protein visualization and analysis
Applications of Western Blotting:
- To figure out how much protein is in a given sample and what size it is.
- Antibody to a virus or bacteria is detected in serum, which aids in disease diagnosis.
- The confirming test for HIV is the Western blotting technique. It looks for anti-HIV antibodies in the patient’s blood.
- It’s a good way to find out if a protein is faulty. Take, for example, Prions illness.
- Creutzfeldt-Jakob disease, Lyme disease, Hepatitis B, and Herpes are all diagnosed with a definitive test.
- determining the amount of post-translational changes,
- verifying protein expression in cloning applications, analyzing protein and biomarker expression levels,