Figure 4:Fluorometric C7/C9 analysis. The PEB and PAB crude samples (100 µg as BSA) were treated with monoSia-specific sialidase, electrophoresed and blotted onto PVDF membrane. PolySia-NCAM at greater than 100 kDa area were cut out and subjected to fluorometric C7/C9 analysis. The samples were treated with mild periodate and non-reducing terminal end of α-2,8-linked oligo/polySia was changed to C7-analogues while internal sialic acid (C9-sialic acid) remain unchanged. After acid treatment, C7- and C9-Sias were released and labeled with DMB. The DMB-labeled sialic acids were applied to the HPLC equipped with a Wako Handy ODS column (250×4.6 mm, Wako), and a fluorescence detector (FP-2025, JASCO). The column is equilibrated using methanol/acetonitrile/water (7:9:84, v/v/v) at 26°C. Samples were applied to HPLC analysis isocratically flow at 1.0 ml/min. (A) Typical chromatograms of C7/C9 analysis of PEB and PAB samples. (B) Relative amounts of C9-Neu5Ac and C7-Neu5Ac derived from PEB and PAB samples. The amount of C9-Neu5Ac derived from PEB was set equal to 1.0. (C) The (C9+C7)/C7 index. The index of PEB was set to 1.0.