 Western blot antibody detection

Blocking occupies unused binding sites on the Western blot membrane. The primary antibody is applied, unbound material is washed away, and a labelled secondary antibody detects retained primary antibody. A fluorescence label emits detectable light after excitation; an enzyme-linked label can instead generate a colored product or light. Washing reduces background but does not prove that remaining binding is selective.

In the original experiment, blocking used excess protein. An anti-ribosome-protein serum followed by labelled secondary antibody produced colored or fluorescence bands; preimmune serum supplied comparison strips. The color readout was an enzyme reaction product, rather than a measurement of target enzyme activity. ^[ref]