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Cross-reactivity occurs when an antibody also recognizes another antigen beyond the one against which it was raised. This may involve related features, but obvious overall similarity is not required. ^[ref]

For an imagined example, a paratope contacts a small surface patch shared by two otherwise different proteins. Both can bind even though their remaining surfaces differ. Increasing antibody concentration can reveal weak binding that was previously undetectable. Conversely, an extra Western blot band need not prove cross-reactivity: the intended protein may occur in multiple forms, or detection reagents may contribute background. The target-deleted and no-primary antibody comparisons under Western blot antibody specificity controls test different explanations.

In an indirect Western blot, the primary antibody binds an epitope on the proposed target protein. Its binding must be tested in the actual sample and assay conditions through Western blot antibody specificity controls.

A secondary antibody recognizes another antibody, rather than the proposed target protein directly. In Western blot antibody detection, its attached label makes retained primary antibodies visible. A secondary antibody must recognize the kind of primary antibody used.

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]

A known target-containing sample is a positive comparison for Western blot antibody detection. A matched sample with gene deletion supplies a negative comparison: disappearance of the proposed band supports the assignment, while a persisting band indicates binding unrelated to the deleted target. Deletion must actually remove the recognized protein; changes in other proteins can complicate the comparison. Omitting the primary antibody checks binding or background from the secondary antibody and detection reagents, but cannot exclude binding of the primary antibody to an unintended protein. Independent antibodies recognizing different epitopes add evidence. ^[ref]

Western blot loading controls ask how much sample entered each lane; Western blot transfer controls ask what reached the receiving sheet; Western blot antibody specificity controls ask what the antibodies recognized. Each closes a different gap in Western blot band interpretation.