Practice question

Membranes used for Western blotting, whether nitrocellulose or PVDF, possess high protein binding capacity and inevitably expose vacant hydrophobic surfaces after antigen transfer. Without intervention, primary antibodies and enzyme-linked secondary antibodies adsorb directly to these empty sites, producing high background and obscuring specific bands. Blocking step saturates unoccupied area with inert proteins such as non-fat dry milk, bovine serum albumin, casein, or irrelevant immunoglobulins, often supplemented with non-ionic detergent Tween-20. This prevents nonspecific antibody capture, enhances signal-to-noise ratio, improves specificity, and enables detection of low-abundance antigens with quantitative reliability.