TITLE: Ion-exchange purification of fucoidans
AUTHORS: Andreas Sichert, Jan-Hendrik Hehemann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Fucoidans are a diverse class of sulfated polysaccharides integral to the cell wall of brown algae and due to their various bioactivities, they are potential drugs. Standardized work with fucoidans is required for structure-function studies, but remains challenging since available fucoidan preparations are often contaminated with other algal compounds. Additionally, fucoidans are structurally diverse depending on species and season, urging the need for standardized purification protocols. Here, we use ion-exchange chromatography to purify different fucoidans and found a high structural diversity between fucoidans. Ion-exchange chromatography efficiently removes the polysaccharides alginate and laminarin and other contaminants such as proteins and phlorotannins across a broad range of fucoidans from major brown algal orders including </span><span style = "font-style:italic;">Ectocarpales</span><span>, </span><span style = "font-style:italic;">Laminarinales</span><span> and </span><span style = "font-style:italic;">Fucales</span><span>. By monomer composition, linkage analysis and NMR characterization, we identified glucuronic acid and O-acetylation as new structural features of certain fucoidans and provide a novel structure of fucoidan from </span><span style = "font-style:italic;">Durvillaea potatorum </span><span>with a-1,3 linked fucose backbone and b-1,6 and b-1,3 galactose branches. This study emphasizes the use of standardized ion-exchange chromatography to obtain defined fucoidans for subsequent molecular studies.</span></div></div></div>

[STEPS]
?. [Buffer preparation]
Filter all buffers through a 0.2 μm bottle top filter to remove dust
?. [Prepare polysaccharide solution]
Mix 0.5 g fucoidan in 500 mL of 50 mM Tris pH 7.5 in ddH2O
?. [Prepare polysaccharide solution]
Use a magnetic stirrer and heat ~50-60°C for ~1h or until fucoidans dissolved
?. [Prepare polysaccharide solution]
Transfer solution into centrifuge vials and spin down at
Centrifuge: 4500 34
?. [Prepare polysaccharide solution]
Carefully decant supernatant into new bottle, avoid transfering solids
?. [Buffer preparation]
~2 liters of double de-ionized water (ddH2O)~2 liters of 50 mM Tris-HCl pH=7.5 in ddH2O (Buffer A)~1 liters of 50 mM Tris-HCl pH=7.5 in ddH2O with 0.5 M NaCl (Buffer B)~1 liters of 50 mM Tris-HCl pH=7.5 in ddH2O with 5 M NaCl (Buffer C)~1 liters of250 mM NaOH in ddH2O
?. [Chromatography]
Set the flow of the pump to 40-50 mL/min
?. [Chromatography]
Equilibrate the column with 3 CV Buffer A
?. [Chromatography]
Apply fucoidan in a semi-batch mode. For this, connect the outlet tube to your input reservoir to circulate the solution at least 3x over the column.
?. [Chromatography]
Wash with 1 CV Buffer A
?. [Chromatography]
Wash with 3 CV Buffer B
?. [Chromatography]
Elute with 0.5 CV Buffer C
?. [Chromatography]
Wash column
?. [Post processing]
Transfer the eluted polysaccharide fraction into a 1 kDa dialysis tubing
?. [Post processing]
Dialyse against ddH2O and change the ddH2O several times
?. [Post processing]
Transfer the liquid into a new vial
?. [Post processing]
Freeze-dry the sample
?. [Chromatography]
2 CV Buffer C
?. [Chromatography]
2 CV ddH2O
?. [Chromatography]
2 CV NaOH
?. [Chromatography]
2 CV ddH2O or until pH is neutral
?. [Chromatography]
Collect the first 100 mL of eluted Buffer C
?. [Chromatography]
Keep in mind the void volumn of your column (~200 mL). After switching to Buffer C, you need to wait one CV. The high salt concetration of Buffer C results in visible schlieren in a low salt buffer.
?. [Chromatography]
Proceed with the eluted fraction to the dialysis
?. [Simple chromatography setup ]
Prepare a simple chromatography system consisting of:MasterFlex L/S peristaltic pump XK50/250 column (GE healthcare)Suitable tubings and connectorsSlurry 200 mL of the ANX and pack it into the columnCompress the resin by applying a gentle flow (~5 mL/min)
?. [Buffer preparation]
Rinse filters with ddH2O and dry for reuse
?. [Chromatography]
For long-term storage of the column, use 20% Ethanol in ddH20