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. 2016 Apr 4;11(4):e0152904. doi: 10.1371/journal.pone.0152904

Fig 2. Purification and identification of prostasin-HAI-1 complex in human milk.

Fig 2

(A) The unbound fraction from the CM-Sepharose was further purified by DEAE-Sepharose chromatography. Fractions 10–21, eluted from the DEAE-Sepharose, were analyzed by Western blot for HAI-1 species. (B) The HAI-1 containing fractions eluted from the DEAE column were subjected to immunoaffinity chromatography using HAI-1 mAb M19-Sepharose. The eluted fraction was analyzed by SDS-PAGE for the protein profile (lane 1). MW stands for molecular weight markers. (C) The HAI-1 containing species from the immunoaffinity column were analyzed by 2-dimensional diagonal gel electrophoresis (gel panels on the left) and the indicated bands subjected to MS/MS-based protein identification (table on the right). First dimension electrophoresis was carried out under non-boiled conditions (upper panel); the second dimension was carried out after heat treating the gel strip sliced from the first dimension gel (lower panel). Six protein bands, as indicated, were sliced out for protein identification. The proteins, the number of peptides, and the relative abundance identified from the 6 bands are summarized in the table on the right.