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. 2014 Jan 9;9(1):e82341. doi: 10.1371/journal.pone.0082341

Figure 5. In silico and in vitro analysis of α, α-T and PA.

Figure 5

A) Homology model obtained using the Modeller and visualized by PyMOL of α-subunit of β-conglycinin, α-T and PA, based on NCBI Conserved Domain Search results with α′ subunit as template (α-helices are shown in blue and β-sheets as dark grey arrows on the structure). B) Surface representation of positive overlapped peptides marked in different colors according to the intensity of the reaction (from pale yellow to red); I and II correspond to opposite sides of the α molecule. Positive peptides localized on the surface of the molecule seem to converge in three regions or putative epitopes. The remaining part of the molecule is represented in transparent spheres showing the secondary structure. C) SDS-PAGE of recombinant PA and PN and immunoblotting developed with 1D5 α-casein-specific mAb and a representative serum of CMA patients. Alpha-s1 casein was used as positive control (Molecular masses are shown on the left as kDa). D) Dot-blot of recombinant treated and untreated PA revealed with a pool of CMA patient sera, α-casein-specific mAb and CMP-specific polyclonal rabbit antiserum. Denaturing conditions consisted on: 0.1M DTT solution, 6M urea or 100°C for 10 minutes. Cas: Alpha-s1 casein.