Western Blot is a popular laboratory technique used to identify specific proteins from a complex mixture. The word "Western" is pronounced as [ˈwɛstərn] and refers to the location of this technique's development, i.e., the Western United States. On the other hand, "Blot" is pronounced as [blɑt] and means the transfer of biological molecules from a gel on to a membrane. The combination of these two terms, "Western Blot," thus represents a method for protein detection and analysis that involves transferring proteins from a polyacrylamide gel to a nitrocellulose membrane.
Western blot is a laboratory technique used in molecular biology and biochemistry to detect and analyze specific proteins within a sample. It is named after its modification of the Southern blot technique, which is used to detect specific DNA sequences.
Western blot involves several steps. Firstly, proteins from a sample are separated based on their molecular weight through gel electrophoresis. The separated proteins are then transferred or "blotted" onto a membrane, typically made of nitrocellulose or polyvinylidene difluoride (PVDF). This transfer ensures that the proteins maintain their spatial arrangement from the gel onto the membrane.
Once transferred, the membrane is blocked with a solution containing proteins like bovine serum albumin (BSA) or nonfat dry milk. This step prevents nonspecific binding and enables specific antibody recognition. After blocking, the membrane is incubated with a primary antibody that specifically recognizes the target protein of interest. The primary antibody binds with the target protein, forming an immunocomplex.
To visualize the presence of the target protein, the membrane is then incubated with a secondary antibody, typically labeled with an enzyme or a fluorophore. The secondary antibody binds to the primary antibody, allowing for detection and quantification of the target protein. This can be achieved through various methods such as chemiluminescence, fluorescence, or colorimetry. The intensity of the resulting signal is directly proportional to the amount of the target protein present in the original sample.
Western blotting is a widely utilized technique in research and diagnostic laboratories. It provides valuable information about protein expression, size, and abundance, allowing scientists to explore various aspects of proteomics and gain insights into disease mechanisms, protein-protein interactions, and signaling pathways.
The term "Western Blot" comes from the combination of two specific methods used in molecular biology: the Southern blot and the Western blot. The Southern blot is named after its developer, Edwin Southern, who created the method in the 1970s. It involves the transfer of DNA molecules from a gel to a blotting membrane and subsequent hybridization with a DNA probe to identify specific DNA sequences.
The Western blot, which was developed as a variation of the Southern blot, was named in contrast to the Southern blot due to the use of protein molecules instead of DNA. The Western blot was introduced by Harriett R. Wallin, initially as the "protein blot" in the late 1970s, and later referred to as the Western blot by W. Neal Burnette in 1981 when the method gained popularity.