The western blot also known as protein immunoblot is a technique used to detect specific proteins in a given sample of biological fluid or tissue homogenate. Western bloting technique uses gel electrophoresis to separate the proteins depending on the polypeptide length or by three dimensional structure of the biologically active protein. Then the separated proteins are transferred into a membrane such as nitrocellulose or PVDF, where specific protein is detected using specific antibodies to the target protein. W. Neal Burnette gave the name western blotting for this technique, but the method was first discovered by George Stark at Stanford.
Sample is prepared from solid tissues using homogenizer. Bacteria, virus or environmental samples are also used as sample or source of protein. Enzyme protease and phosphatase inhibitors are added to prevent protein digestion from these enzymes. Tissue preparations are done at cold temperature, to maintain the structure of proteins.
Proteins which are present in the sample are separated using gel electrophoresis. Proteins are separated depending on their isoelectric point, molecular weight, charge or combination of all factors. Gel electrophoresis uses polyacrylamide gels and buffer along with sodium dodecyl sulfate (SDS-PAGE). SDS-PAGE separates protein by their molecular weight, as they remove secondary and tertiary structure of proteins. Greater the concentration of acrylamide, better is the resolution of low molecular weight proteins.
Separated proteins are transferred into a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Membrane is placed on top of the gel with separated proteins along with stack of filter papers. This entire set up is placed in a buffer solution, by the capillary action proteins from the gel are transferred into the membrane. Protein transferring can also be done using electric current and the technique is called as electroblotting. Blocking of non specific binding of protein is done using low concentration of protein solutions, like BSA (Bovine Serum Albumin) is used.
Protein of interest is detected using specific antibody which is linked to a reporter enzyme, these when exposed to enzyme specific substrate produces a colored product. Analysis of the western blot or presence of protein of interest can be done using many techniques, such as Colorimetric method, Chemiluminescent detection, Radioactive detection, Fluorescent detection and also secondary probing method is also used. Radioactive detection has the danger of inducing mutations in the body cells of researcher or investigator. Among all the detection techniques fluorescent detection method is most sensitive detection method for analyzing the specific proteins which are separated by western blot technique.
Applications of Western Blotting in Medical Diagnostics:
1. The confirmatory HIV test uses western blot technique to detect anti-HIV antibody in a serum sample of human.
2. Western Blotting technique is also used in the definitive test for Bovin spongiform encephalopathy, also commonly known as mad cow disease.
3. Western blotting technique is also used to detect some forms of Lyme diseases
4. Confirmatory test for Hepatitis B infection is also done using western blot technique
5. Western blot technique is also used in veterinary medicine to detect and confirm the infection or diseases. For example this technique is used to confirm the FIV+ status in cats.
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