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sugar binding column and deglycosylation

James Fethiere James.Fethiere at BRI.NRC.CA
Thu Sep 28 02:17:52 EST 1995


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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