35 kU/l. Self-prepared cattle allergen mix To detect misclassification or misidentification of sensitized individuals, we additionally applied extracts from the hair of different cattle races found in typical working environments. These additional tests were performed in individuals EPZ5676 who either had work-related symptoms or had at least one positive reaction in one or both of the commercial cattle allergen tests. The hair of purebred adult cattle was obtained from

different breeders throughout Germany. Cattle selected for this study were all healthy to avoid a possible influence of pathology on the Bos d 2 production. The hair was cut close to the skin without visible contamination. The hair of these cattle breeds was used because they were most relevant to allergies: German Brown, Holstein–Friesian, Charolais, Jersey/White-blue Belgian, German Red Pied, Blonde

Aquitaine, and German Simmental (Heutelbeck et al. 2009). About 0.3 g of hair of each individual cow was incubated for different time periods (2 h up to 48 h) at 6°C in 2 ml of a 0.1 M ammonium hydrocarbonate (NH4HCO3) solution. An incubation period of 24 h was found to yield optimal results in protein content and SDS-PAGE separation. The extracts were lyophilized and reconstituted in NH4HCO3. We verified that the lyophilized extracts did not show any differences in total protein content or SDS-PAGE separation compared to the unlyophilized extracts (data not shown). Protein content Saracatinib was determined using the bicinchonic acid procedure (Pierce Chemicals, Rockford, USA). The results were verified using several dilutions of each sample. Proteins were separated using SDS-PAGE. A 14% separating gel (“SERVA-Gel TM

TG 14-Vertical Tris–Glycine Gel”, SERVA, Heidelberg, Germany) was used for performing Coomassie staining of the separated cattle allergen mix, and 15% separating gel (self-prepared) for the immunoblot experiments. Molecular weights (MW) were estimated by comparison with commercial MW standard mixtures (“SERVA Prestained SDS-PAGE Protein check details marker 6.5–200 kDa, Liquid Mix” (Immunoblot), “SERVA Unstained SDS-PAGE Protein Marker 6.5–200 kDa, Liquid Mix” (Coomassie) not SERVA, Heidelberg, Germany). Equal amounts of proteins concentrated at 2 mg/ml for immunoblotting were applied to the polyacrylamide gel electrophoresis, which was conducted at a constant voltage (150 V) for 90–100 min. The marker protein preparations were run alongside the extract. For the investigation of the protein patterns, the gels were stained with Coomassie blue. The molecular weights of the corresponding allergens were estimated relative to the standard marker proteins. Each extract was investigated in an independent immunoblot experiment. Detection of allergens (immunoblotting) The detection of the allergenic proteins in the extracts was performed by immunoblotting.