Hello all,
I have expressed a glycoprotein in sf9 cells, and it seems to be highly
sialated (according to sensitivity to neuraminidase). I am now looking
for a lectin column with a higher specificity and affinity for sialated
carbohydrates as an extra step in my purification scheme. Any suggestions??
The protein migrates as 4 distinct bands on an SDS-PAGE gel. The highest MW
band is the first coming out of the Q-sepharose column, and is also the
one that collapses upon treatment with neuraminidase. However subsequent
treatment of the sample with endo-F did not yield further processing of
the carbohydrates while treatment with N-glycanase/Endo-F alone provoked
collapsing of all 4 bands to a single one. I was wondering why Endo-F
did not have any effect either by itself or after neuraminidase. The enzyme
was not too old, and an excess of it was added ( > 2U/mg protein), so ...
Thank you for your help
../James
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National Research Council of Canada E-mail:James.Fethiere at BRI.NRC.ca
Biotechnology Research Institute office: (514) 496-6173
Macromolecular Crystallography group lab : (514) 496-6376
6100 Royalmount Avenue fax : (514) 496-5143
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