Western blotting

Principle:

The Western blotting (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. In brief, the sample undergoes protein denaturation, followed by gel electrophoresis. A synthetic or animal derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.

Western blotting Procedure/Steps:

1. Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.

Step I: Extraction of Protein

• Use different samples to extract protein(e.g. tissues or cells)
• Use homogenizer or sonication to breakdown samples.
• Prevent sample digestion at cold temperature through protease and phosphates.
• Finally, observe concentration of proteins and use spectrophotometer for protein concentration.

Step II: Gel electrophoresis

• The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide gel electrophoresis.
• The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors.
• The nature of the separation depends on the treatment of the sample and the nature of the gel.

Step III: Blotting

• make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF).
• The most commonly used method for transferring the proteins is called electroblotting. Electroblotting uses an electric current to pull the negatively charged proteins from the gel towards the positively charged anode, and into the PVDF or NC membrane.
• The proteins move from within the gel onto the membrane.

Step IV: Blocking

• Blocking is very important step in western blotting.
• Antibodies are also protein so they are likely to bind the nitrocellulose paper. So before adding the primary antibody the membrane is non-specifically saturated or masked by using casein or Bovine serum albumin (BSA).

Step V: Treatment with Primary Antibody

• Particular proteins are then detected using antibody.
• The specific interaction between the antibody and its antigen occurs on the membrane, and the position of the bound antibody is detected.
• The primary antibody is specific to desired protein so it form Ag-Ab complex.

Step VI: Treatment with secondary antibody

• The Labelled secondary antibody is then added to detect the location of primary antibody.
• The antibody sandwich results in an amplification of the signal.
• The Secondary antibody is often labbeled with an enzyme whose activity in the presence of appropriate substrates results in either a color change on the membrane or the emission of light that can be detected using X-ray film.
• The secondary antibody is enzyme labelled. For eg. alkaline phosphatase or Horseradish peroxidase (HRP) is labelled with secondary antibody.
• Secondary antibody is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab complex.

Step VII: Treatment with suitable substrate

• To visualize the enzyme action, the reaction mixture is incubated with specific substrate.
• The enzyme convert the substrate to give visible colored product, so band of color can be visualized in the membrane.
• Western blotting is also a quantitative test to determine the amount of protein in sample.

Western blotting Application:

• It is most sensitive and specific test for determining size and amount of protein present in any material.
• The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample.
• To determine the size and amount of protein in given sample.
• Disease diagnosis: detects antibody against virus or bacteria in serum.
• Western blotting technique is the confirmatory test for HIV. It detects anti HIV antibody in patient’s serum.
• Useful to detect defective proteins. For eg Prions disease.
• Definitive test for Creutzfeldt-Jacob disease, Lyme disease, Hepatitis B and Herpes

Advantage and Disadvantages

Advantages

• Effective early diagnostic tool.
• Detect minimal immunogenic response form virus or bacteria.
• Requires fewer antibodies for testing.
• Detect specific protein from a large mixture of different proteins. (Even more than 300,000)

Disadvantages

• Requires specific primary antibodies to perform test on desired protein of interest.
• Challenging and hence requires well trained staffs.
• Poorer results as antibodies may revel off-target bindings.
• Detecting and imaging the results can be expensive as equipment cost is high.