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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Options<br />
Product Spacer Coupling conditions Maximum Comments<br />
arm<br />
operating flow<br />
CNBr-activated None pH 7–9, 2–16 hours, 400 cm/h* Supplied as a freeze-<br />
Sepharose 4 Fast Flow +4 °C - room temp. dried powder.<br />
CNBr-activated None pH 8–10, 2–16 hours, 75 cm/h* Supplied as a freeze-<br />
Sepharose 4B +4 °C - room temp. dried 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 />
There are many examples in the literature <strong>of</strong> the use <strong>of</strong> CNBr-activated Sepharose.<br />
Figure 61 shows the separation <strong>of</strong> a native outer envelope glycoprotein, gp120, from HIV-1<br />
infected T-cells. Galanthus nivalis agglutinin (GNA), a lectin from the snowdrop bulb, was<br />
coupled to CNBr-activated Sepharose 4 Fast Flow to create a suitable affinity medium.<br />
A 280 nm<br />
0.5<br />
native gp120<br />
0<br />
24 hours 40 min Time<br />
Fig. 61. Separation <strong>of</strong> native gp120 protein on GNA coupled to CNBr-activated Sepharose 4 Fast Flow.<br />
From Gilljam, G. et al., Purification <strong>of</strong> native gp120 from HIV-1 infected T-cells. Poster presented at Recovery <strong>of</strong><br />
Biological Products VII, Sept. 25-30, 1994, San Diego, CA, USA. Further details are available in the CNBr-activated<br />
Sepharose 4 Fast Flow datafile, from Amersham Pharmacia Biotech.<br />
Buffer preparation<br />
Acidification solution: 1 mM HCl (kept on ice)<br />
Coupling buffer: 0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3<br />
Blocking buffer: 1 M ethanolamine or 0.2 M glycine, pH 8.0<br />
Wash buffer: 0.1 M acetate, 0.5 M NaCl, pH 4<br />
Preparation <strong>of</strong> CNBr-activated Sepharose 4 Fast Flow <strong>and</strong> CNBr-activated Sepharose 4B<br />
1. Suspend the required amount <strong>of</strong> freeze-dried powder in ice-cold 1 mM HCl (HCl preserves the activity <strong>of</strong> the<br />
reactive groups that hydrolyze at high pH).<br />
2. Wash for 15 min. on a sintered glass filter (porosity G3), using a total <strong>of</strong> 200 ml 1 mM HCl per gram dry<br />
powder, added <strong>and</strong> sucked <strong>of</strong>f in several aliquots. The final aliquot <strong>of</strong> 1 mM HCl is sucked <strong>of</strong>f until cracks<br />
appear in the cake.<br />
3. Transfer the matrix immediately to the lig<strong>and</strong> solution.<br />
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