ORIGINAL PAPER Identification of proteins in painting cross-sections by immunofluorescence microscopy M. Vagnini & L. Pitzurra & L. Cartechini & C. Miliani & B. G. Brunetti & A. Sgamellotti Received: 28 December 2007 / Revised: 27 February 2008 / Accepted: 29 February 2008 / Published online: 8 April 2008 # Springer-Verlag 2008 Abstract Immunofluorescence microscopy offers a highly specific analytical tool for unambiguous recognition and mapping of proteins in complex matrices. In the present work, the analytical potentials of immunofluorescence microscopy have been exploited to provide recognition of proteinaceous binders in painting cross-sections. An optimised analytical protocol is proposed for the identification of ovalbumin and of bovine serum albumin as markers of egg white and casein, respectively. The study has been carried out on laboratory model samples simulating both easel and mural paintings. The obtained results demonstrated the effectiveness of the method, suggesting the potential future use of immunofluorescence microscopy as a routine diagnostic tool in conservation science. Possible developments of the proposed methodology in order to improve the specificity of the method and its detection sensitivity are presented and discussed. Keywords Immunofluorescence microscopy . Proteinaceous binder . Painting cross-section Introduction Proteinaceous substances are widely encountered within the large class of natural organic products that have been used as raw materials in works of art. Animal glue, egg, milk or casein have been widely used as binding media or adhesives in paintings and polychrome sculptures due to their ability to form adherent and elastic films of high durability [1, 2]. Furthermore, the inherent adhesive properties of these compounds have also favoured their diffusion into conservation treatments, making it more difficult to distinguish between original and added materials found in works of art. Over the last decade a lot of attention has been paid to the development of new analytical approaches aimed at providing a full characterisation of artwork materials in a totally noninvasive or microdestructive way. However, the identification of organic materials is still a challenging task due to the wide variety of compounds historically used in artistic production, and because they are usually charac- terised by complex mixtures of different chemical species. They exhibit a more pronounced tendency to undergo degradation processes than inorganic components [2], and this complicates their recognition. Many efforts have been undertaken to develop combined analytical procedures capable of providing detailed information on the organic components used in artistic and historical works; these are mainly based on the use of spectroscopic, chromatographic and mass-spectrometric techniques or capillary zone elec- trophoresis [3–9]. Recent technological developments in scientific instru- mentation have offered new opportunities for creating either nondestructive or microdestructive techniques with improved performance in terms of spatial resolution [4, 8, 10–14]. In spite of the increased amount of information Anal Bioanal Chem (2008) 392:57–64 DOI 10.1007/s00216-008-2041-9 M. Vagnini (*) : B. G. Brunetti : A. Sgamellotti Centro SMAArt, Dipartimento di Chimica, Università di Perugia, via Elce di Sotto 8, 06123 Perugia, Italy e-mail: manuela@thch.unipg.it L. Pitzurra Centro SMAArt, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Perugia, Via del Giochetto, 06123 Perugia, Italy L. Cartechini : C. Miliani : A. Sgamellotti Istituto di Scienze e Tecnologie Molecolari, CNR, c/o Dipartimento di Chimica via Elce di Sotto 8, 06123 Perugia, Italy