32.13:
Western Blotting
Western blotting is an analytical technique used to detect the protein of interest from a protein mixture.
It can help identify protein isoforms and post-translational protein modifications. Further, it can ascertain target proteins following drug treatment.
This method builds on the SDS-PAGE that separates proteins based on the charge to mass ratio.
These proteins are transferred to a nitrocellulose membrane, typically under an electric current.
The negatively charged proteins move towards the anode and are transferred to the membrane retaining their original position.
The membrane is then incubated with a primary antibody, specific to the protein of interest. A prior incubation in a blocking buffer prevents the binding of antibodies to the membrane.
Next, the secondary antibody is tagged with reporter enzymes, like horseradish peroxidase or a fluorophore, which binds to the primary antibody.
When incubated with an appropriate substrate, these reporter enzymes produce color or light, or emit fluorescent signals allowing the detection of the specific protein in the mixture.
32.13:
Western Blotting
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The proteins from the polyacrylamide gel are transferred to specialized membranes like nitrocellulose (NC) or polyvinylidene difluoride (PVDF). Compared to NC, PVDF has higher durability and protein binding capacity and can be re-probed.
The protein transfer is either by electric current application (electroblotting) or capillary action. In electroblotting, the hydrophobic interactions transfer the negatively charged proteins from the gel and trap them on the membrane. In the other method, a filter paper placed on the gel soaks the buffer by capillary action, simultaneously drawing the proteins from the gel and transferring them to the membrane. To ensure the quality of transferred bands, non-specific protein stains like Ponceau S allow for a reversible check.
The next step, immunoblotting, involves treating the membrane with a blocking buffer like 3-5% Bovine serum albumin or non-fat dried milk. These buffers reduce the chance of non-specific binding of the primary antibody to the membrane. The primary antibodies can either be polyclonal or monoclonal and bind to the protein bands on the membrane through their Fab region. Next, the added secondary antibodies bind and attach to the Fc region of the primary antibodies. The secondary antibodies are tagged with enzymes which, upon suitable substrate addition, produce detectable colorimetric or chemiluminescent signals. Further, densitometry techniques can quantify the optical density of these imaged bands, corresponding to the abundance of proteins in the sample.
The expected result for a western blot can be affected by various factors like erroneous sample loading, inaccurate transfer with trapped air bubbles, and the use of non-specific antibodies. Also, inappropriate primary and secondary antibody concentrations and contaminated buffers can affect the quality of bands and their visualization.
Suggested Reading
- Jensen, E. C.(2012). The Basics of Western Blotting. American Association for Anatomy. 295 (3) : 369-371.https://doi.org/10.1002/ar.22424
- Kurien, B. T., & Scofield, R. H. (2015). Western blotting: an introduction. Methods in molecular biology (Clifton, N.J.), 1312, 17–30. https://doi.org/10.1007/978-1-4939-2694-7_5