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Figure 1 Methanolic Homogenate / Three-Phase Separation Lower Phase Upper Phase Interface/ Interface/ CMW Insoluble CMW Soluble DEAE-cellulose AG1-X2 DEAE-cellulose (optional) nuc-sugars bisphosphates free glycans M2Gn2 – G3M9Gn2-PP-Dol man-P-Dol glc-P-Dol Gn1 – M1Gn2-PP-Dol hexose-1-P hexose-6-P glycoproteins N-glycanase weak acid weak acid M2Gn2 – G3M9Gn2 free glycans mannose glucose Gn1 – M1Gn2 hexoses hexose-6-P hexoses hexose-6-P free N-glycans AMAC labeling/ monosaccharide gel: ++ + + (Gn1) ANDS labeling/ oligosaccharide gel: ++ ++ (Gn2 –M1Gn2)

Figure 2 M M M M +H2O; Son.; Cent. +CH3OH; Son. Recovery of CH3OH-soluble material Dry CH3OHSusp. #2 CH3OH Sup. Aq. Sup. + Pellet Res. Sup. Susp. P P P P P P +CHCl3; Son.; Cent. Interface +10:10:3; Son. Cent.; Decant U Cent. Dry Int. L CH3OHSusp. #1 MoistPellet Three Phases 10:10:3 Susp. 10:10:3 Sup. + Pellet Protein Pellet Recover Liq. Phases; Dry Recovered Liq. Phase U L Dry Apply directly to DEAE-Cellulose if additional purification is needed OR Fractions in RED tubes can be discarded if only LLOs are needed Upper (Highly Polar) Lower (Highly Apolar) LLO

Figure 3 Recovered 10:10:3 Liq. Phase (LLO) Dry Directly Apply To DEAE-Cellulose Column OR 20 vol. 10:10:3 wash (neutral impurities) 10 vol. 10:10:3 / 3 mM NH4OAc (MPD, GPD traces) 10 vol. 10:10:3 / 300 mM NH4OAc (LLO) Dry Eluate #3 Redissolve in 10:10:3 and Dry (x2) to remove NH4OAc Hydrolysis: 0.1 N HCl/isopropanol/50 oC/1 hr. Dry Partition: n-butanol/water Collect lower water phase Dry LLO-derived Glycans

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