## 3 Results and Discussion
### Analysis of Binding Media and Wood Specimens
#### 3.2.2 Analysis of Paint Sample: Egg, Animal Adhesive, and Casein (Proteins)  
GC-MS analysis identified amino acids in all specimens, although the amount varies from sample to sample. Hydroxproline, which is indicative of animal glue, was found in all specimens (Table S16, see Supplementary Material). Additionally, glycine was the most abundant amino acid for all samples except for those from H1d and H3a, and one of the samples from E1d. These results are also suggestive of an animal-based glue, as glycine is the most abundant amino acid found in animal glues, whereas aspartic acid and serine, and glutamic acid and leucine are most abundant in egg and casein, respectively [48]. Most samples closely match the amino acids profile of animal glue, with a correlation coefficient ranging from 0.93-1.00 (Table 4). The two samples with the lowest correlation were the four layered sample from H1c and the two layered sample with insect from E1d, which resulted in correlation coefficients of 0.75 and 0.37, respectively. If a paint specimen is comprised of a single binding medium (e.g., only bone glue) the correlation coefficient would be equal to 1.0, although values greater than 0.97 are considered acceptable [22]. However, a significant reduction in the correlation coefficient occurs if the test specimen is composed of multiple proteins [26]. Here, the addition of the consolidant convolutes the interpretation of the amino acid analysis. Previous analysis performed on specimens from Nore, Rollag, and Uvdal (triangles in Figure 1) confirmed that an animal-based adhesive was used as the main binding material, with occasional modifications [3]. At Nore and Uvdal, egg was identified in some of the blue pigmented areas, whereas casein was identified in the ground layer of Nore, Rollag, and Uvdal [3]. However, neither casein nor egg have been confirmed through additional analysis. Furthermore, casein used as a binding medium was not recorded in Norway until ca. 1750 [3], which is a little over a century after the polychrome paintings of Heddal and Eidsborg were rendered. Therefore, it is not surprising that neither egg nor casein were identified during this study. Table 4 also reports the per cent protein obtained from the test specimens. No significant difference was observed between the protein content of the two sites, nor was a trend obtained within the three different sample categories. While the per cent protein of the specimen from Heddal ranged between 1.7 and 10.8%, the samples from Eidsborg were  
composed of 2.2-9.7% protein (when excluding E1d). Additionally, when comparing the average amount of protein within the white ground layer (basecoat), similar amounts were obtained (Heddal = 5.6 + 1.7% (n = 4), Eidsborg 5.4 + 2.2% (n = 5)). ELISA confirmed the presence of fish glue in all specimens (Table 4). See Supplementary Material for a summary of the absorbance values (Figure S9, see Supplementary Material). Most of the samples were strongly positive for fish glue (above 0.45 OD) and mammal glue was not detected (below 0.3 OD). Only two of the samples (ground layer of H1c and paint layers of H2e) resulted in a weak positive for fish (around 0.4 OD). Absorbance intensity is directly correlated to the concentration of proteinaceous glue and can be affected by degradation (loss of tertiary structure) or cross linking with pigments. In addition, egg and casein antibodies were not utilized in this study and mixtures of proteins would be difficult to detect with GC-MS. Amino acid analysis is not always accurate, especially when mixtures of proteins are present because after hydrolysis, amino acids combine and lose their peptide signature. As such, the identification of egg and animal glue mixtures, based on amino acid analysis, could be misleading. Proteomics or immunological techniques are preferable for the identification of mixtures and can also be used for species identification. Lastly, most of the FT-IR spectra displayed weak absorption peaks related to animal glue (Figure S6 and Table S14, see Supplementary Material).