Pa glucose oligomer

The sialidase-sensitive glycans obtained by anion-exchange HPLC, as well as the neutral glycans, were further separated by a dual gradient revered-phase HPLC analysis using an Inertsil ODS-3 column (2.1 φ × 150 mm; GL Sciences) (36 (link)). Briefly, the flow rate was 0.2 ml/min at 25 °C, and eluent A (0.1 M ammonium acetate buffer, pH 6.4) and eluent B (0.1 M ammonium acetate buffer, pH 4.0 with 0.5% 1-butanol) was used; the gradient program (expressed as % eluent B) was as follows: 0 to 10 min, isocratic 1%; 10 to 110 min, 1%-70%; 110.1 to 120 min, isocratic 70%; and 120.1 to 150 min, isocratic 1%. The fluorescence was detected at an emission wavelength of 400 nm with an excitation wavelength of 320 nm. The presence of sialic acid was further confirmed using A. ureafaciens sialidase (Roche Applied Science). A 2,3-Specific sialidase (GLYKO Sialidase S from S. pneumoniae, ProZyme) was used for the linkage analysis of sialic acid. For neutral glycans, the occurrence of high mannose-type glycans was confirmed by treatment with jack bean α-mannosidase (Seikagaku Kogyo Co). Each fraction was quantitated using the peak area relative to the standard PA-glucose hexamer in the PA-glucose oligomer (2 pmol/μl; Takara Bio, Inc) as a reference.