1 / 3

Figure 1

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.

gilead
Download Presentation

Figure 1

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. 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)

  2. 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

  3. 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

More Related