Dpa 6s column

MDMΦ FOS were detected as previously described by Alonzi et al. [22 (link)]. Cells were cultured in 6-well plates as described above, and protein lysates were prepared according to the freeze/thaw protocol. Following cell lysis, samples were subjected to mixed-bed ion exchange and then lyophilised. FOS were labelled with 2-aminobenzoic acid (2-AA) and purified using a DPA-6S column (Sigma). Unconjugated 2-AA was removed by phase splitting with ethyl acetate, and samples were lyophilised and resuspended in water and then purified using a con-A column. Glycans were separated by normal phase-high performance liquid chromatography (NP-HPLC), and peak area was used to assess molar quantity in comparison to standards of known identity and quantity. FOS generation was normalised to protein concentration based on a modified Bradford assay as previously described [35 (link)]. Briefly, cell lysates were added in a 1:1 volumetric ratio to 1x Bradford Quick Start Reagent (Bio-Rad) and incubated at 20°C for 5 minutes. Absorbance values (A_xxx) at 595 nm and 420 nm were measured, and the ratio of A₅₉₅ to A₄₂₀ was used to determine protein concentration in comparison to a serially diluted standard of bovine serum albumin.

Post mortem liver samples were assayed for the presence of free oligosaccharides (FOS) as previously described by Alonzi et al [41 (link)]. Around 30mg (wet weight) of liver was lysed by cycles of freeze-thawing in double-distilled water. Following cell lysis, samples were subjected to mixed-bed ion exchange and then lyophilised. FOS were labelled with 2-aminobenzoic acid (2-AA) and purified using a DPA-6S column (Sigma). Unconjugated 2-AA was removed by phase splitting with ethyl acetate, and samples were lyophilised and resuspended in water then purified using a concanavalin-A column. Glycans were separated by normal phase-high performance liquid chromatography (NP-HPLC) and peak area was used to assess molar quantity in comparison to standards of known identity and quantity. FOS generation was normalised to wet weight.