Tuft protein is a material associated with enamel tufts, and resides in dental enamel primarily at the enamel–dentine junction. It is located primarily at prism peripheries and extends in a very attenuated form towards the enamel outer surface. While it appears to be a mixture of components, partial sequencing and antibody studies have demonstrated the presence of amelin, a protein associated with prism boundaries. Biochemical investigations have been seriously hampered by the fact that tuft protein is extremely insoluble in a range of solvents, including mineral acids, EDTA, chaotropic agents, and detergents including SDS. This raised the question as to whether it could be chemically cross-linked. Antibodies to γ-glutamyl cross-linking peptide were used to determine the presence of a cross-linking isopeptide. In all cases examined, a positive response indicated that tuft protein does display chemical cross-linking, which may explain the insoluble nature of this material. This may be a mechanism to prevent protein degradation at the enamel–dentine junction during the degradation of enamel matrix, which occurs during amelogenesis.