Highprep 16 60 sephacryl s 300 hr column

Delipidated vesicle and matrix pellets containing carbohydrates and proteins were washed twice with acetone, dried under a stream of nitrogen, and reconstituted in 3 ml of 20 mM bis-Tris/HCl (pH 6.5) loading buffer. Aliquots were chromatographically desalted on a HiPrep 26/10 Desalting column (GE Healthcare Life Sciences, Uppsala, Sweden) and then separated on an anion exchanger HiPrep 16/10 DEAE FF column (GE Healthcare Life Sciences) equilibrated with 20 mM bis-Tris/HCl (pH 6.5). Carbohydrate positive flow-through fractions were pooled together, lyophilized, resuspended in 15% acetonitrile in 150 mM ammonium bicarbonate, and applied to gel filtration on a HighPrep 16/60 Sephacryl S-300 HR column (GE Healthcare). All chromatographic separation steps were performed at room temperature on the high-performance liquid chromatography ÄKTA-Purifier 10 system (GE Healthcare Life Sciences).

Additional structural analysis was performed after a series of purification and fractionation steps to further ascertain the presence of a mannan-glucan complex. These steps include size exclusion chromatography followed by separation on an anion exchanger HiPrep 16/10 DEAE FF column (GE Healthcare Life Sciences). Neutral free carbohydrates were collected in flowthrough fractions, which were pooled and applied to gel filtration on a HighPrep 16/60 Sephacryl S-300 HR column (GE Healthcare Life Sciences), yielding 22 individual polysaccharide peaks, F1 to F22. The molecular weight of biofilm matrix neutral carbohydrates was determined using size exclusion column calibration with a set of Leuconostoc species dextran standards (polymers with molecular weights [in thousands] of 100, 70, 40, 25, and 6). Both high-molecular-weight (HMW) and low-molecular-weight (LMW) fractions were examined by GC for monosugar analysis. Matrix monosugar composition and quantification was performed on alditol acetate derivatives by GLC-FID (Shimadzu GC-2010 system; Shimadzu, Kyoto, Japan).