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WGA coupling with CNBr-activated sepharose 4B


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#1 ebitaq

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Posted 04 March 2005 - 05:28 AM

I want to constract WGA-sepharose 4B by CNBr activation. However, some precipitates were observed after addition of WGA solution to CNBr-activated sepharose 4B. Absorbance of WGA solution decreased a fifth compared with those before addition to the sepharose. I thought that the precipitates was WGA because the precitates appeared as soon as WGA solution was added. But, Why?
WGA was soluble in PBS (PH8.3). CNBr-activated sepharose was equilibrated with 10 column volume of 0.2M NaHCO3 (PH8.3) after reaction with CNBr in acetonitril. PH changes presumably cause the precipitaion.
Please give me some advises.

#2 quol

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Posted 04 March 2005 - 06:06 AM

I used CNBr activated Sepharose 4B a lot and I had once the problem you described.
If the concentration of the ligand you want to couple is high, the handling must be carefull. Otherwise it aggregates.
To avoid such precipitates, I incubate the protein wiith Sepharose in the tube with gentle rotation end-over-end, having as little air as possible in the tube.
This should help




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