Western immunoblotting is a common laboratory technique used to detect specific proteins in a sample. The spelling of the word can be broken down into three parts: "west-ern," "im-mu-no-" and "-blot-ting." The first part uses the /w/ sound followed by the diphthong /ɛə/, spelled with "e" and "r." The second part has the /ɪ/ sound, spelled with "i," followed by the /m/ sound, and the stressed /nu/ sound spelled with "o." The final part has the /blɑt/ sound, spelled with "blot," followed by the /ɪŋ/ ending.
Western immunoblotting, also known as Western blot or Western blot analysis, is a laboratory technique used to detect specific proteins in a sample. This method is often employed in molecular biology and biochemistry research to analyze protein expression levels and identify specific protein targets.
The process of Western immunoblotting involves several steps. First, a sample containing proteins of interest is separated based on their molecular weight using gel electrophoresis, typically polyacrylamide gel electrophoresis (PAGE). The proteins are then transferred, or "blotted," from the gel onto a solid support such as a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. This transfer ensures that the proteins maintain their spatial arrangement based on size.
Once the proteins are on the membrane, they are probed with specific antibodies against the protein targets of interest. The antibodies specifically bind to their respective protein targets, allowing for their detection. These antibodies are typically labeled with a secondary antibody that is conjugated to an enzyme or a fluorescent or radioactive tag. This secondary antibody binds to the primary antibody, amplifying the signal and making visualization of the bound proteins possible.
The final step involves developing the Western blot image through a chemical reaction that produces a signal in the presence of the conjugated enzyme or tag. The resulting bands or spots on the membrane correspond to the presence and size of the proteins of interest. By comparing the protein bands in the sample to known standards or controls, the relative quantity of specific proteins can be determined.
Western immunoblotting is a versatile technique widely used in research and diagnostic applications. It allows for the identification and quantification of target proteins in complex samples, aiding in the understanding of cellular and molecular processes in various disciplines, including molecular biology, immun
The term "Western Immunoblotting" is derived from the combination of various concepts and techniques.
1. Western: In biochemistry and molecular biology, the term "Western" refers to the technique of transferring proteins separated by gel electrophoresis onto a solid support such as a membrane. This technique was developed by physicist Edwin Southern in the 1970s as an extension of the earlier Southern blotting technique used for DNA analysis.
2. Immunoblotting: Immunoblotting is a specific type of Western blotting that involves the use of antibodies to detect specific proteins. It combines the principles of Western blotting with immunological methods to analyze protein samples. The term "immuno-" in immunoblotting indicates the involvement of antibodies for protein detection.