Biotin NTA (BNTA) is a bifunctional molecule consisting of nitrilotriacetic acid conjugated to biotin. The NTA moiety chelates a Ni(II) ion at four of its six coordination sites, leaving two sites available for coordination with histidine residues. This interaction enables selective binding to polyhistidine-tagged proteins immobilized on nitrocellulose membranes. Detection limits of approximately 0.1 pmol have been reported under standard blotting conditions.

The biotin group allows detection through enzyme-linked streptavidin systems such as streptavidin–horseradish peroxidase conjugates, as well as fluorescent avidin derivatives. Binding of the NTA–Ni(II) complex to His-tagged proteins is reversible and can be disrupted at pH ~4.8, permitting removal of the probe and subsequent reanalysis of the blot with alternative detection reagents. This reagent is commonly used for detecting polyhistidine-containing biomolecules, including recombinant fusion proteins.