James Fethiere (James.Fethiere at BRI.NRC.CA) wrote:
: 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??
I would try SNA (specific for a(2-6) SA). This works well for terminal
sialic acids in this linkage, which yours are likely to be given your CHO
seems to be an N-linked structure.
: 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 ...
Endo F cleaves between the two GlcNAc moieties whilst PNGaseF
(N-glycanase) cleaves between Asp and sugar. It is a much better enzyme
for this sort of thing.
SNA is Sambucus Nigra agglutinin. The buffer I use is 50mM Tris/1mM
CaCl2/1mM MgCl2, pH 7.4 (HCl). I elute bound material using this buffer
with 0.1M Lactose (odd choice but it works, neat sialic acid is far too
expensive to use as a competeing ligand. SNA can be obtained from Boehringer.
Hope this helps.
--
__________________________________________________
Steven Pirie-Shepherd
srps at galactose.mc.duke.edu
"Insert your own pithy phrase just about here!"
