Journal: Scientific Reports

Article Title: Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages

doi: 10.1038/srep16800

Figure Lengend Snippet: ( a ) HPLC analysis of oligoSia with a degree of polymerization of 6 (oligoSia DP6) and polySia with average degree of polymerization 18, 20 and 22 (polySia avDP18, avDP20 and avDP22) compared to hydrolyzed polySia. DMB-labeled oligoSia DP6, polySia avDP18, avDP20 and avDP22 were eluted by a NaCl buffer gradient. The HPLC retention times were monitored. ( b ) Polyacrylamide gel electrophoresis of polySia avDP18, avDP20 and polySia avDP22 visualized by stains-all solution. PolySia avDP20 showed a molecular weight of ~6 kDa. Dextran sulfate with molecular weights of ~3 kDa and ~7 kDa served as molecular weight controls. Representative image out of at least three independent experiments is shown. ( c ) Polyacrylamide gel electrophoresis of biotinylated polySia avDP20 (Biot. av DP20) visualized by stains-all solution. Majority of biotinylated polySia avDP20 is still intact after biotinylation. Representative image out of at least three independent experiments is shown. ( d ) ELISA to demonstrate the interaction between polySia with avDP20 and SIGLEC-11. SIGLEC-11 Fc-fusion protein was coated to an ELISA plate. Biotinylated polySia avDP20 (0.01, 0.05, 0.25, 1.25 and 6.25 μg/ml) or biotinylated dextran (0.01, 0.05, 0.25, 1.25 and 6.25 μg/ml) were added and detected by HRP conjugated streptavidin, followed by a TMB reaction. PolySia avDP20 bound to SIGLEC-11 Fc-fusion protein, while there was negligible binding of biotinylated dextran. Data are presented as relative binding (values of OD 450 measurements) mean +/− SEM of n = 3 independent experiments. ***p < 0.001; ANOVA followed by Bonferroni.

Article Snippet: Afterwards, blocking solution was removed and different concentrations of biotinylated-polySia avDP20 (0.01, 0.05, 0.25, 1.25, 6.25 μg/ml) or biotinylated dextran (5 kDa, NANOCS) serving as control (0.01, 0.05, 0.25, 1.25, 6.25 μg/ml diluted in PBS) were added and incubated for 2 hours.

Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Molecular Weight, Enzyme-linked Immunosorbent Assay, Binding Assay