Sidestepping the challenge of casein quantification in ancient paintings by dot-blot immunoassay

A straightforward procedure based on dot-blot immunoassay is proposed as an effective diagnostic tool suitable for detecting and quantifying milk casein in cultural heritage samples. A polyclonal primary antibody, denaturing conditions and the standard addition method were used to overcome barriers common to the traditional analysis of protein-based artistic materials, providing the possibility of achieving specific and detailed results in an easy and cost effective way. The optimized procedure detected, and successfully quantified, casein in both freshly dried and artificially aged model samples prepared with milk casein and various pigments (azurite, calcite, cinnabar, minium and red ochre). Moreover, the experiments revealed that pigments as well as artificial ageing did not differently influence the dot-blot response. Thermogravimetric analysis confirmed the obtained results. The detection and quantification of casein applied to canvas 70-years ago for conservation purposes provided final proof of the feasibility of the methodology. Despite the ageing, the complex matrix and the micro-size of the sample, it was possible to detect and quantify casein by dot-blot immunoassay. The specific and unambiguous result makes the proposed protocol a suitable procedure to recognize aged proteins with a degraded amino acid pattern.