What is the role of the primary antibody in immunoblotting?

The primary antibody in immunoblotting serves the critical function of specifically binding to the protein of interest. This specificity is crucial for identifying and quantifying that particular protein among the multitude of proteins present in a complex mixture. When a sample is subjected to gel electrophoresis and subsequently transferred to a membrane, the primary antibody recognizes a unique epitope—or binding site—on the target protein.

This binding enables the researchers to isolate and visualize the presence of the protein using secondary antibodies that are conjugated to a detectable marker, such as an enzyme or a fluorescent dye. The specificity of the primary antibody ensures that only the protein of interest is targeted during this process, making it a fundamental component in the success of the immunoblotting technique.

The other options do not accurately represent the role of the primary antibody: blocking agents (as mentioned in the first choice) are used to reduce background noise by saturating potential non-specific binding sites, visualization of all proteins (as in the third choice) is typically accomplished using a general protein stain, and the separation of proteins (as in the fourth choice) is performed during the gel electrophoresis step prior to the application of antibodies.