Analytical and Bioanalytical Chemistry

, Volume 408, Issue 1, pp 203–215 | Cite as

Influence of pigments and protein aging on protein identification in historically representative casein-based paints using enzyme-linked immunosorbent assay

  • Fang Ren
  • Natalya Atlasevich
  • Brian Baade
  • John Loike
  • Julie ArslanogluEmail author
Research Paper


A systematic study on the influence of pigments and sample aging on casein identification was performed on 30 reconstructed paints. The protein in all the paints was extracted into solution for analysis. The amount of protein that can be retrieved for solution-based analysis in each of the reconstructed paints was studied with a well-developed NanoOrange method before and after artificial aging. The results showed that in the paints with calcium phosphate (in bone black) and copper carbonate, hydroxide, or acetate (in verdigris and azurite), the amount of protein that can be retrieved for liquid-phase analysis is much smaller than the other paints, indicating that the protein degradation was accelerated significantly in those paints. Carbon (in vine black), calcium carbonate (in natural chalk), and calcium sulfate (terra alba gypsum and ground alabaster) did not affect much the amount of protein that can be retrieved in the paints compared to non-pigmented binder, meaning that the protein degradation rate was not affected much by those pigments. Artificial aging was observed to decrease the amount of retrievable protein in all the reconstructed paints that were studied. The enzyme-linked immunosorbent assay (ELISA) method was applied to the 28 reconstructed paints (except two verdigris paints) to assess the protein identification. The ELISA responses from the different paints were compared at fixed protein concentrations. Natural chalk, bone black, raw sienna, stack lead white, and cochineal red-violet lake had the lowest ELISA signal in this study, which indicated that the binding sites (epitopes) on the target protein in these paints are likely to deteriorate more than those in the other paints. Artificial aging did not influence the ELISA response as much as the pigments when the protein concentration was kept the same for the paints that were studied.


Casein paint Cultural heritage Protein degradation Pigment and protein interaction ELISA NanoOrange 



The authors gratefully acknowledge financial support from National Science Foundation CHE-1041839. We thank Ms. M. Herre from the Rockefeller University for reproducing the NanoOrange and ELISA data. We also thank A. Sturm and J. DeLuca from University of Delaware for their contribution in making reconstructed paints.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2015_9089_MOESM1_ESM.pdf (289 kb)
ESM 1 (PDF 289 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Fang Ren
    • 1
  • Natalya Atlasevich
    • 1
    • 4
  • Brian Baade
    • 2
  • John Loike
    • 3
  • Julie Arslanoglu
    • 1
    Email author
  1. 1.Department of Scientific ResearchThe Metropolitan Museum of ArtNew YorkUSA
  2. 2.Art ConservationUniversity of DelawareNewarkUSA
  3. 3.College of Physicians and SurgeonsColumbia UniversityNew YorkUSA
  4. 4.Genzyme Science CenterFraminghamUSA

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