Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation
Fig 4
Chromatographic purification of the pro-inflammatory activity present in BALF-SEA.
(A) BALFs were obtained from mice 16 h after i.n. SEA or BSS, concentrated using a 3 kDa centricon and loaded on a size exclusion column (Sephacryl 100-HR). Protein detection was recorded at 280 nm (top panel) and fractions from BALF-SEA and BALF-BSS were tested in the bioassay (bottom panel). BALF-SEA fractions with bioactivity were pooled and concentrated on centricon 3 kDa while the corresponding fractions from BALF-BSS were processed identically. Each sample run was a pool of BALF obtained from 3–5 mice. Representative of 1 out 5 experiments is shown. (B) Samples were concentrated, the buffer was exchanged on a PD-10 column to 20 mM Phosphate pH 7.0, applied to an anion exchange column (High Q) and an elution profile was developed with a linear gradient of 0–1 M NaCl. Protein detection was recorded at 280 nm (top panel), bound and unbound fractions were collected, tested in a bioassay (bottom panel), and pooled as described in A. Representative of 1 out 4 experiments is shown. (C) Samples were concentrated, buffer exchanged on a PD-10 column to 20 mM MES pH 5.5, applied to a cation exchange column (High S) and an elution profile was developed with a linear gradient of 0–1 M NaCl. Protein detection was recorded (top panel), bound and unbound fractions collected, tested (bottom panel) and pooled as described in A. Representative of 1 out 3 experiments is shown.