Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
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Coupling through hydroxy, amino or thiol groups<br />
via a 12-carbon spacer arm<br />
Epoxy-activated Sepharose 6B<br />
Epoxy-activated Sepharose 6B is used for coupling lig<strong>and</strong>s that contain hydroxyl, amino or<br />
thiol groups. Because <strong>of</strong> the long hydrophilic spacer arm, it is particularly useful for coupling<br />
small lig<strong>and</strong>s such as choline, ethanolamine <strong>and</strong> sugars. The pre-activated matrix is formed<br />
by reacting Sepharose 6B with the bis oxirane, 1,4 bis-(2,3-epoxypropoxy-)butane. The<br />
partial structure is shown in Figure 66.<br />
S<br />
epharose<br />
O CH2 CH CH2 O (CH 2)<br />
4 O CH2<br />
CH CH2<br />
OH<br />
O<br />
Fig. 66. Partial structure <strong>of</strong> Epoxy-activated Sepharose 6B.<br />
A stable ether linkage is formed between the hydrophilic spacer <strong>and</strong> the matrix. Free oxirane<br />
groups couple via stable ether bonds with hydroxyl-containing molecules such as sugars,<br />
via alkylamine linkages with lig<strong>and</strong>s containing amino groups, <strong>and</strong> via thioether linkages<br />
with lig<strong>and</strong>s containing thiol groups.<br />
Options<br />
Product Spacer Substitution Coupling conditions Maximum operating Comments<br />
arm per ml matrix flow<br />
Epoxy-activated 12-atom 19–40 µmoles pH 9–13, 16 hours - 75 cm/h* Supplied as a<br />
Sepharose 6B epoxy groups several days, freeze-dried<br />
+20 - +40 °C powder.<br />
*See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from<br />
measurement in a packed column with a bed height <strong>of</strong> 10 cm <strong>and</strong> i.d. <strong>of</strong> 5 cm.<br />
Purification example<br />
A 280 nm<br />
Fucose-specific lectin<br />
Elution volume<br />
Fig. 67. <strong>Chromatography</strong> <strong>of</strong> a crude extract <strong>of</strong> Ulex europaeus on fucose coupled to Epoxy-activated Sepharose 6B,<br />
column volume 11 ml. Extract was applied in 0.9% NaCl. Fucose-specific lectin was eluted with 5 ml fucose (50 mg/ml).<br />
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