The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein. A secondary antibody is added which recognizes and binds to the primary antibody. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off.
A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination. The western blot (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. Analytical technique used in molecular biology Western blot workflow
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